Methods and Systems for Methylotrophic Production of Organic Compounds

ABSTRACT

The present disclosure identifies pathways, mechanisms, systems and methods to confer production of carbon-based products of interest, such as sugars, alcohols, chemicals, amino acids, polymers, fatty acids and their derivatives, hydrocarbons, isoprenoids, and intermediates thereof, in engineered and/or evolved methylotrophs such that these organisms efficiently convert C1 compounds, such as formate, formic acid, formaldehyde or methanol, to organic carbon-based products of interest, and in particular the use of organisms for the commercial production of various carbon-based products of interest.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 61/734,472 filed Dec. 7, 2012, the disclosure of whichis incorporated herein by its entirety.

STATEMENT REGARDING GOVERNMENT LICENSE RIGHTS

This invention was made with government support under contract numberDE-AR0000091 awarded by U.S. Department of Energy, Office of ARPA-E. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

The invention relates to systems, mechanisms and methods to conferproduction of carbon-based products to a methylotroph or amethylotrophic organism to efficiently convert C1 compounds into variouscarbon-based products, and in particular the use of such organism forthe commercial production of various carbon-based products of interest.The invention also relates to systems, mechanisms and methods to conferadditional and/or alternative pathways for energy conversion,methylotrophy and/or carbon fixation to a methylotroph.

BACKGROUND

Heterotrophs are biological organisms that utilize energy from organiccompounds for growth and reproduction. Commercial production of variouscarbon-based products of interest generally relies on heterotrophicorganisms that ferment sugar from crop biomass such as corn or sugarcaneas their energy and carbon source [Bai, 2008]. An alternative tofermentation-based bio-production is the production of carbon-basedproducts of interest from photosynthetic organisms, such as plants,algae and cyanobacteria, that derive their energy from sunlight andtheir carbon from carbon dioxide to support growth [U.S. Pat. No.7,981,647]. However, the algae-based production of carbon-based productsof interest relies on the relatively inefficient process ofphotosynthesis to supply the energy needed for production of organiccompounds from carbon dioxide [Larkum, 2010]. Moreover, commercialproduction of carbon-based products of interest using photosyntheticorganisms relies on reliable and consistent exposure to light to achievethe high productivities needed for economic feasibility; hence,photobioreactor design remains a significant technical challenge[Morweiser, 2010].

Methylotrophs are biological organisms that utilize energy and/or carbonfrom C1 compounds containing no carbon-carbon bonds such as formate,formic acid, formaldehyde, methanol, methane, halogenated methanes, andmethylated sulfur species to produce all multi-carbon, organic compoundsnecessary for growth and reproduction. Most existing, naturally-occuringmethylotrophs are poorly suited for industrial bio-processing and havetherefore not demonstrated commercial viability for this purpose. Suchorganisms have long doubling times relative to industrializedheterotrophic organisms such as Escherichia coli, reflective of lowtotal productivities. In addition, techniques for genetic manipulation(homologous recombination, transformation or transfection of nucleicacid molecules, and recombinant gene expression) are inefficient,time-consuming, laborious or non-existent.

Thus, a need exists to develop engineered and/or evolved methylotropssuitable for industrial uses. Accordingly, the ability to endow amethylotroph with biosynthetic capability to produce carbon-basedproducts of interest, to grow the engineered and/or evolved methylotrophat the high cell densities needed for industrial bio-processing, and toefficiently provide the engineered organism with C1 compounds wouldsignificantly enable more energy- and carbon-efficient production ofcarbon-based products of interest. In addition, the ability to add oneor more additional or alternative pathways for energy conversion,methylotrophy and/or carbon fixation capability to the methylotrophwould enhance its ability to produce carbon-based products on interest.

SUMMARY

Systems and methods of the present invention provide for efficientproduction of renewable energy and other carbon-based products ofinterest (e.g., fuels, sugars, chemicals) from C1 compounds.Furthermore, systems and methods of the present invention can be used inthe place of traditional methods of producing chemicals such as olefins(e.g., ethylene, propylene), which are traditionally derived frompetroleum in a process that generates toxic by-products that arerecognized as hazardous waste pollutants and harmful to the environment.As such, the present invention can additionally avoid the use ofpetroleum and the generation of such toxic by-products, and thusmaterially enhances the quality of the environment by contributing tothe maintenance of basic life-sustaining natural elements such as air,water and/or soil by avoiding the generation of hazardous wastepollutants in the form of petroleum-derived by-products in theproduction of various chemicals.

In certain aspect, the invention described herein provides amethylotroph engineered to confer biosynthetic production of variouscarbon-based products of interest from C1 compounds. The engineeredorganism comprises one or more at least partially engineered carbonproduct biosynthetic pathways that convert central metabolites intodesired products, such as carbon-based products of interest.Carbon-based products of interest include but are not limited toalcohols, fatty acids, fatty acid derivatives, fatty alcohols, fattyacid esters, wax esters, hydrocarbons, alkanes, polymers, fuels,commodity chemicals, specialty chemicals, carotenoids, isoprenoids,sugars, sugar phosphates, central metabolites, pharmaceuticals andpharmaceutical intermediates. For example, the carbon-based products ofinterest can include one or more of a sugar (for example, glucose,fructose, sucrose, xylose, lactose, maltose, pentose, rhamnose,galactose or arabinose), sugar phosphate (for example,glucose-6-phosphate or fructose-6-phosphate), sugar alcohol (forexample, sorbitol), sugar derivative (for example, ascorbate), alcohol(for example, ethanol, propanol, isopropanol or butanol), fermentativeproduct (for example, ethanol, butanol, lactic acid, lactose oracetate), ethylene, propylene, 1-butene, 1,3-butadiene, acrylic acid,fatty acid (for example, ω-cyclic fatty acid), fatty acid intermediateor derivative (for example, fatty acid alcohol, fatty acid ester,alkane, olegin or halogenated fatty acid), amino acid or intermediate(for example, lysine, glutamate, aspartate, shikimate, chorismate,phenylalanine, tyrosine, tryptophan), phenylpropanoid, isoprenoid (forexample, hemiterpene, monoterpene, sesquiterpene, triterpene,tetraterpene, polyterpene, isoprene, bisabolene, myrcene,amorpha-4,11-diene, farnesene, taxadiene, squalene, lanosterol,β-carotene, ζ-carotene, lycopene, phytoene, limonene, or polyisoprene),glycerol, 1,3-propanediol, 1,4-butanediol, 1,3-butadiene,polyhydroxyalkanoate, polyhydroxybutyrate, lysine, γ-valerolactone, andacrylate. In some embodiments, the carbon-based products of interest canbe carbon-based central metabolites.

The resulting engineered and/or evolved methylotroph of the invention iscapable of efficiently synthesizing carbon-based products of interestfrom C1 compounds. The invention also provides carbon productbiosynthetic pathways for conferring biosynthetic production of thecarbon-based product of interest upon the host organism where theorganism lacks the ability to efficiently produce carbon-based productsof interest from C1 compounds. The invention also provides methods forintroducing the carbon product biosynthetic pathways into themethylotroph. The invention also provides methods and media compositionsfor culturing the engineered and/or evolved methylotroph to supportefficient methylotrophic production of carbon-based products ofinterest.

In various embodiments, the invention provides for the C1 compoundserving as a source of both energy and carbon for the organism. In oneembodiment, the C1 compound is soluble or miscible in water. Forexample, the C1 compound can be one or more of formate, formic acid,methanol and/or formaldehyde. C1 compounds that dissolve at highconcentration or are miscible in water, in some instances, arepreferable to less soluble or immiscible chemical species, such asmethane, because mass transfer and uptake by the organism is moreefficient. Similarly, soluble C1 compounds are preferable to molecularhydrogen, carbon dioxide or carbon monoxide, used in autotrophicproduction of carbon-based compounds (see, e.g., Example 7). In someembodiments, the C1 compound can be soluble in other solvents thanwater, depending on the composition of the media used for growing theorganism. For example, the solubility of the C1 compound in the mediamay be enhanced by other components therein. In some embodiments, the C1compound can be derived from electrolysis.

In certain embodiments, one or more of the following carbon productbiosynthetic pathways can be used:

-   -   when said carbon product biosynthetic pathway is for fatty acid        biosynthesis, said carbon product biosynthetic pathway includes        one or more of: fatty acid synthase, acetyl-CoA carboxylase,        fatty-acyl-CoA reductase, aldehyde decarbonylase, lipase,        thioesterase and acyl-CoA synthase peptides; or    -   when said carbon product biosynthetic pathway is for branched        chain fatty acid biosynthesis, said carbon product biosynthetic        pathway includes one or more of: branched chain amino acid        aminotransferase, branched chain α-ketoacid dehydrogenase,        dihydrolipoyl dehydrogenase, beta-ketoacyl-ACP synthase,        crotonyl-CoA reductase, isobutyryl-CoA mutase, β-ketoacyl-ACP        synthase I, trans-2,cis-3-decenoyl-ACP isomerase and        trans-2-enoyl-ACP reductase II; or    -   when said carbon product biosynthetic pathway is fatty alcohol        biosynthesis, said carbon product biosynthetic pathway includes        one or more of: fatty alcohol forming acyl-CoA reductase, fatty        alcohol forming acyl-CoA reductase, alcohol dehydrogenase and        alcohol reductase; or    -   when said carbon product biosynthetic pathway is for fatty ester        biosynthesis, said carbon product biosynthetic pathway includes        one or more of: alcohol O-acetyltransferase, wax synthase, fatty        acid elongase, acyl-CoA reductase, acyltransferase, fatty acyl        transferase, diacylglycerol acyltransferase, acyl-CoA was        alcohol acyltransferase, bifunctional wax ester        synthase/acyl-CoA:diacylglycerol acyltransferase, and        β-ketoacyl-ACP synthase I; or    -   when said carbon product biosynthetic pathway is for alkane        biosynthesis, said carbon product biosynthetic pathway includes        one or more of: decarbonylase and terminal alcohol        oxidoreductase; or    -   when said carbon product biosynthetic pathway is for ω-cyclic        fatty acid biosynthesis, said carbon product biosynthetic        pathway includes one or more of: 1-cyclohexenylcarbonyl CoA        reductase, 5-enopyruvylshikimate-3-phosphate synthase, acyl-CoA        dehydrogenase, enoyl-(ACP) reductase, 2,4-dienoyl-CoA reductase,        and acyl-CoA isomerase; or    -   when said carbon product biosynthetic pathway is for halogenated        fatty acid biosynthesis, said carbon product biosynthetic        pathway includes one or more of: fluorinase, nucleotide        phosphorylase, fluorometabolite-specific aldolase,        fluoroacetaldehyde dehydrogenase, and fluoroacetyl-CoA synthase;        or    -   when said carbon product biosynthetic pathway is the        deoxylylulose 5-phosphate (DXP) isoprenoid pathway, said carbon        product biosynthetic pathway includes one or more of:        1-deoxy-D-xylulose-5-phosphate synthase,        1-deoxy-D-xylulose-5-phosphate reductoisomerase,        4-diphosphocytidyl-2C-methyl-D-erythritol synthase,        4-diphosphocytidyl-2C-methyl-D-erythritol kinase,        2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase,        (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase,        isopentyl/dimethylallyl diphosphate synthase and        4-hydroxy-3-methylbut-2-enyl diphosphate reductase; or    -   when said carbon product biosynthetic pathway is the        mevalonate-dependent (MEV) isoprenoid pathway, said carbon        product biosynthetic pathway includes one or more of: acetyl-CoA        thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate        kinase, phosphomevalonate kinase, mevalonate pyrophosphate        decarboxylase and isopentenyl pyrophosphate isomerase; or    -   when said carbon product biosynthetic pathway is the        glycerol/1,3-propanediol biosynthesis pathway, said carbon        product biosynthetic pathway includes one or more of:        sn-glycerol-3-P dehydrogenase, sn-glycerol-3-phosphatase,        glycerol dehydratase and 1,3-propanediol oxidoreductase; or    -   when said carbon product biosynthetic pathway is the        1,4-butanediol/1,3-butadiene biosynthesis pathway, said carbon        product biosynthetic pathway includes one or more of:        succinyl-CoA dehydrogenase, 4-hydroxybutyrate dehydrogenase,        aldehyde dehydrogenase, 1,3-propanediol oxidoreductase and        alcohol dehydratase; or    -   when said carbon product biosynthetic pathway is the        polyhydroxybutyrate biosynthesis pathway, said carbon product        biosynthetic pathway includes one or more of:        acetyl-CoA:acetyl-CoA C-acetyltransferase,        (R)-3-hydroxyacyl-CoA:NADP⁺ oxidoreductase and        polyhydroxyalkanoate synthase; or    -   when said carbon product biosynthetic pathway is the lysine        biosynthesis pathway, said carbon product biosynthetic pathway        includes one or more of: aspartate aminotransferase, aspartate        kinase, aspartate semialdehyde dehydrogenase,        dihydrodipicolinate synthase, dihydrodipicolinate reductase,        tetrahydrodipicolinate succinylase,        N-succinyldiaminopimelate-aminotransferase,        N-succinyl-L-diaminopimelate desuccinylase, diaminopimelate        epimerase, diaminopimelate decarboxylase, L,L-diaminopimelate        aminotransferase, homocitrate synthase, homoaconitase,        homoisocitrate dehydrogenase, 2-aminoadipate transaminase,        2-aminoadipate reductase, aminoadipate semialdehyde-glutamate        reductase and lysine-2-oxoglutarate reductase; or when said        carbon product biosynthetic pathway is the chorismate        biosynthesis pathway, said carbon product biosynthetic pathway        includes one or more of: 2-dehydro-3-deoxyphosphoheptonate        aldolase, 3-dehydroquinate synthase, 3-dehydroquinate        dehydratase, NADPH-dependent shikimate dehydrogenase,        NAD(P)H-dependent shikimate dehydrogenase, shikimate kinase,        3-phosphoshikimate-1-carboxyvinyltransferase and chorismate        synthase; or    -   when said carbon product biosynthetic pathway is the        phenylalanine biosynthesis pathway, said carbon product        biosynthetic pathway includes one or more of: chorismate mutase,        prephenate dehydratase and phenylalanine transaminase; or    -   when said carbon product biosynthetic pathway is the tyrosine        biosynthesis pathway, said carbon product biosynthetic pathway        includes one or more of: chorismate mutase, prephenate        dehydrogeanse and tyrosine aminotransferase; or    -   when said carbon product biosynthetic pathway is the        γ-valerolactone biosynthesis pathway, said carbon product        biosynthetic pathway includes one or more of: propionyl-CoA        synthase, beta-ketothiolase, acetoacetyl-CoA reductase,        3-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoA Δ-isomerase,        4-hydroxybutyryl-CoA transferase and 1,4-lactonase; or    -   when said carbon product biosynthetic pathway is the butanol        biosynthesis pathway, said carbon product biosynthetic pathway        includes one or more of: beta-ketothiolase, acetoacetyl-CoA        reductase, 3-hydroxybutyryl-CoA dehydrogenase, enoyl-CoA        hydratase, butyryl-CoA dehydrogenase, trans-enoyl-coenzyme A        reductase, butyrate CoA-transferase, aldehyde dehydrogenase,        alcohol dehydrogenase, acetyl-CoA acetyltransferase,        β-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl CoA        dehydrogenase, CoA-acylating aldehyde dehydrogenase and        aldehyde-alcohol dehydrogenase; or    -   when said carbon product biosynthetic pathway is the acrylate        biosynthesis pathway, said carbon product biosynthetic pathway        includes one or more of: enoyl-CoA hydratase, propionyl-CoA        synthase and acrylate CoA-transferase.

In some embodiments, the 1-deoxy-D-xylulose-5-phosphate synthase can beencoded by SEQ ID NO:1, or a homolog thereof having at least 80%sequence identity; or the isopentenyl pyrophosphate isomerase can beencoded by SEQ ID NO:2, or a homolog thereof having at least 80%sequence identity. In one embodiment, when said carbon productbiosynthetic pathway is the isoprene biosynthesis pathway, said carbonproduct biosynthetic pathway can include isoprene synthase. The isoprenesynthase can be encoded by SEQ ID NO:3, or a homolog thereof having atleast 80% sequence identity. In another embodiment, when said carbonproduct biosynthetic pathway is the bisabolene biosynthesis pathway,said carbon product biosynthetic pathway can include E-alpha-bisabolenesynthase. The E-alpha-bisabolene synthase can be encoded by SEQ ID NO:4,or a homolog thereof having at least 80% sequence identity.

In certain embodiments, the methylotrophic organism can be selected fromthe class Alphaproteobacterium. The methylotrophic organism may also beselected from the genus Paracoccus. For example, the methylotrophicorganism can be Paracoccus denitrificans, Paracoccus versutus orParacoccus zeaxanthinifaciens.

In some embodiments, the engineered cell can be further modified to havea less reduced growth rate on electrolytically generated C1 compoundrelative to non-evolved methylotrophic organism, or a substantiallysimilar or enhanced growth rate on electrolytically generated C1compound relative to non-electrolytically generated C1 compound. Incertain embodiments, the engineered cell can be further evolved to havea less reduced growth rate on electrolytically generated C1 compoundrelative to non-evolved methylotrophic organism, or a substantiallysimilar or enhanced growth rate on electrolytically generated C1compound relative to non-electrolytically generated C1 compound.

In another aspect, an evolved methylotrophic organism is provided,having a less reduced growth rate on electrolytically generated C1compound relative to non-evolved methylotrophic organism, or having asubstantially similar or enhanced growth rate on electrolyticallygenerated C1 compound relative to non-electrolytically generated C1compound.

In a further aspect, a method for selecting an evolved methylotrophicorganism having improved growth on a C1 compound is provided,comprising: incubating methylotrophic cells in a culture chamber withcontrolled temperature, cell concentration, and medium inflow andoutflow rates, wherein the medium inflow includes a C1 compound;continuously monitoring a concentration of biomass in the culturechamber; and adjusting a flow rate of the C1 compound into the culturechamber so as to continually maintain an environment that selects for animproved growth rate. In some embodiments, the method can furtherinclude adjusting the medium inflow to be more permissive of growth ormore suppressive of growth, so as to provide an adaptive environment toselect for a fitness of the cells. In certain examples, the C1 compoundcan be formate. The C1 compound may be electrolytically generated. TheC1 compound can be soluble in water.

In yet another aspect, a method of introducing a conjugative plasmidinto methylotrophic host cells is provided, comprising: incubating amixture of predetermined ratios of a donor culture and a recipientculture, at temperatures between 4° C. and 37° C. for between 1 and 48hours, wherein the donor culture comprises a conjugal donor containing aconjugative plasmid having a first selectable trait, and the recipientculture comprises methylotrophic host cell having a second selectabletrait; and subjecting the incubated mixture to a dually selectivecondition where only plasmid-containing transconjugants that have boththe first selectable trait and the second selectable trait can grow,wherein the method does not include centrifugation or filtration of themixture or incubated mixture. In some embodiments, the conjugal donorcan be an E. coli strain such as E. coli S17-1, or an E. coli harboringplasmids such as pRK2013 or pRK2073, or any E. coli strain expressing atra operon capable of mobilizing plasmids containing an RP4-derivedsequence. The conjugal donor can be in a different species or genus ofthe host cell. In certain embodiments, the transconjugated plasmidcontains an RP4 or similar mob element. In some embodiments, the hostcell can be from the class Alphaproteobacterium or from the genusParacoccus. For example, the host cell can be Paracoccus denitrificans,Paracoccus versutus or Paracoccus zeaxanthinifaciens.

A further aspect of the invention relates to a composition for bacterialculture, formulated to provide formate as the sole source of C1 compoundand to enhance the growth of methylotrophic bacteria. The compositioncan contain between 0 and 160 mM sodium bicarbonate, between 0 and 16 mMsodium chloride, between 0 and 100 mM sodium nitrate, between 0 and 30mM sodium thio sulfate, and initially containing between 5 and 100 mM ofa formate salt, such as sodium formate or ammonium formate. For example,the composition can contain 100 mM sodium bicarbonate, 6 mM sodiumchloride, 6 mM sodium nitrate, 11 mM sodium thiosulfate, and 26 mMsodium formate or ammonium formate. The composition can further includea basal minimal medium. In some embodiments, the basal minimal mediumcan be MOPS minimal medium, M9 minimal medium, R medium, M63 medium, ora medium substantially similar to any of the foregoing.

In yet another aspect, a composition of bacterial culture is provided,which is formulated to provide formate as the sole C1 compound and toenhance the growth of methylotrophic bacteria in a fed-batch bioreactor.The composition can include a medium initially charged in the fed-batchbioreactor which comprises R medium supplemented with between 1 and 100micromolar sodium molybdate, between 10 and 1000 nanomolar sodiumselenite, between 0.01 to 1 mg/L of thiamine, and between 0.001 to 1mg/L of cobalamin. For example, the medium can contain between 5 and 20micromolar sodium molbydate, between 50 and 200 nanomolar sodiumselenite, between 0.05 to 2 mg/L of thiamine, and between 0.01 and 0.2mg/L cobalamin. The composition can further include a feed compositionsupplied to the fed-batch bioreactor comprising a formate salt atsupramolar concentration. The formate salt can be ammonium formateand/or sodium formate. The feed composition can further include asupramolar concentration of nitrate salt such as sodium nitrate. Thenitrate salt and the formate salt can be provided in a molar ratio of3.2:8, 3.0:8 or lower.

Also provided herein is a method for culturing methylotrophic bacteria,comprising incubating methylotrophic bacteria in any of the compositionsdescribed herein. In some embodiments, the incubating can be conductedaerobically. The incubating can take place in a fed-batch bioreactor. Insome embodiments, a volumetric rate of C1 feedstock consumption in thefed-batch reactor can exceed 1.5 g*L⁻¹ hr⁻¹. In certain embodiments, theincubating can be conducted in the presense of a nitrate salt aselectron acceptor and with a C1 feedstock as electron donor. In someembodiments, the C1 feedstock is a formate salt, such as sodium formateor ammonium formate. During incubation, the molar ratio of the nitratesalt to the formate salt can be kept below 3.2:8. The nitrate salt andthe formate salt can be provided to the fed-batch bioreactor in a feedcomposition in supramolar concentrations in a molar ratio of 3.0:8 orlower. In some embodiments, the formate salt can be ammonium formateand/or sodium formate. The nitrate salt can be sodium nitrate.

In some aspects, growth of the methylotroph on C1 compounds can beaugmented by the addition of additional and/or alternative pathways forenergy conversion, methylotrophy and/or carbon fixation. Exemplaryenergy conversion pathways and carbon fixation pathways are described inU.S. Pat. No. 8,349,587, the entirety of which is hereby incorporatedherein by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the metabolic reactions of the ribulose monophosphatecycle [Strom, 1974]. Some methylotrophic organisms such as Methylococcuscapsulatus use this formaldehyde assimilation pathway to make thecentral metabolites needed for growth. In metabolite names, —P denotesphosphate. Each reaction is numbered. Enzymes catalyzing each reactionare as follows: 1, hexulose-6-phosphate synthase (E.C. 4.1.2.43);2,6-phospho-3-hexuloisomerase (E.C. 5.3.1.27); 3, phosphofructokinase(E.C. 2.7.1.11); 4, fructose bisphosphate aldolase (E.C. 4.1.2.13); 5,transketolase (E.C. 2.2.1.1); 6, transaldolase (E.C. 2.2.1.2); 7,transketolase (E.C. 2.2.1.1); 8, ribose 5-phosphate isomerase (E.C.5.3.1.6); 9, ribulose-5-phosphate-3-epimerase (E.C. 5.1.3.1).

FIG. 2 depicts the metabolic reactions of the serine cycle. Somemethyltrophic organisms such as Hyphomicrobium methylovorum GM2,Hyphomicrobium zavarzinii ZV580, Methylobacterium extorquens AM1,Methylobacterium organophilum, Methylocystis parvus, Methylosinussporium and Methylosinus trichosporium use this formaldehydeassimilation pathway to make the central metabolites needed for growth.In metabolite names, —P denotes phosphate and -CoA denotes coenzyme A.Enzymes catalyzing each reaction are as follows: 1, spontaneousreaction; 2, serine hydroxymethyltransferase (E.C. 2.1.2.1); 3,serine-glyoxylate aminotransferase (E.C. 2.6.1.45); 4, hydroxypyruvatereductase (E.C. 1.1.1.81); 5, glycerate 2-kinase (E.C. 2.7.1.165); 6,enolase (E.C. 4.2.1.11); 7, phosphoenolpyruvate carboxylase (E.C.4.1.1.31); 8, malate dehydrogenase (E.C. 1.1.1.37); 9, malate thiokinase(E.C. 6.2.1.9); 10, malyl-CoA lyase (E.C. 4.1.3.24); 11, the glyoxylateregeneration pathway.

FIG. 3 depicts the metabolic reactions of energy conversion pathway(s)that oxidize C1 compounds in some methylotrophic organisms such asParacoccus species. Each reaction is numbered. Enzymes catalyzing eachreaction are as follows: 1, methanol dehydrogenase (E.C. 1.1.2.7); 2,methylamine dehydrogenase (E.C. 1.4.9.1); 3, S-(hydroxymethyl)glutathione synthase (E.C. 4.4.1.22); 4, NAD- and glutathione-dependentformaldehyde dehydrogenase (E.C. 1.1.1.284); 5, S-formylglutathionehydrolase (E.C. 3.1.2.12); 6, formate dehydrogenase (E.C. 1.2.1.2).

FIG. 4 depicts the metabolic reactions of the Calvin-Benson-Basshamcycle or the reductive pentose phosphate (RPP) cycle [Bassham, 1954].Some methylotrophic organisms such as Paracoccus species use this carbonfixation pathway to reduce carbon dioxide to central metabolites neededfor growth. In metabolite names, —P denotes phosphate. Each reaction isnumbered. Enzymes catalyzing each reaction are as follows: 1, ribulosebisphosphate carboxylase (E.C. 4.1.1.39); 2, phosphoglycerate kinase(E.C. 2.7.2.3); 3, glyceraldehyde-3P dehydrogenase (phosphorylating)(E.C. 1.2.1.12 or E.C. 1.2.1.13); 4, triose-phosphate isomerase (E.C.5.3.1.1); 5, fructose-bisphosphate aldolase (E.C. 4.1.2.13); 6,fructose-bisphosphatase (E.C. 3.1.3.11); 7, transketolase (E.C.2.2.1.1); 8, sedoheptulose-1,7-bisphosphate aldolase (E.C. 4.1.2.-); 9,sedoheptulose bisphosphatase (E.C. 3.1.3.37); 10, transketolase (E.C.2.2.1.1); 11, ribose-5-phosphate isomerase (E.C. 5.3.1.6); 12,ribulose-5-phosphate-3-epimerase (E.C. 5.1.3.1); 13, phosphoribulokinase(E.C. 2.7.1.19).

FIG. 5 depicts the metabolic reactions of the reductive tricarboxylicacid cycle [Evans, 1966; Buchanan, 1990; Hiigler, 2011]. Somemethylotrophic organisms such as Nautilia sp. strain AmN use this carbonfixation pathway to reduce carbon dioxide to central metabolites neededfor growth. Each reaction is numbered. For certain reactions, such asreaction 1 and 7, there are two possible routes denoted by a and b, eachof which is catalyzed by different enzyme(s). Enzymes catalyzing eachreaction are as follows: 1a, ATP citrate lyase (E.C. 2.3.3.8); 1b,citryl-CoA synthetase (E.C. 6.2.1.18) and citryl-CoA lyase (E.C.4.1.3.34); 2, malate dehydrogenase (E.C. 1.1.1.37); 3, fumaratedehydratase or fumarase (E.C. 4.2.1.2); 4, fumarate reductase (E.C.1.3.99.1); 5, succinyl-CoA synthetase (E.C. 6.2.1.5); 6, 2-oxoglutaratesynthase or 2-oxoglutarate:ferredoxin oxidoreductase (E.C. 1.2.7.3); 7a,isocitrate dehydrogenase (E.C. 1.1.1.41 or E.C. 1.1.1.42); 7b,2-oxoglutarate carboxylase (E.C. 6.4.1.7) and oxalosuccinate reductase(E.C. 1.1.1.41); 8, aconitate hydratrase (E.C. 4.2.1.3); 9, pyruvatesynthase or pyruvate:ferredoxin oxidoreductase (E.C. 1.2.7.1); 10,phosphoenolpyruvate synthetase (E.C. 2.7.9.2); 11, phosphoenolpyruvatecarboxylase (E.C. 4.1.1.31).

FIG. 6 is a block diagram of a computing architecture.

FIG. 7 provides a schematic to convert succinate or 3-hydroxypropionateto various chemicals.

FIG. 8 provides a schematic of glutamate or itaconic acid conversion tovarious chemicals.

FIG. 9 depicts the metabolic reactions of a galactose biosyntheticpathway. In metabolite names, —P denotes phosphate. Each reaction isnumbered. Enzymes catalyzing each reaction are as follows: 1,alpha-D-glucose-6-phosphate ketol-isomerase (E.C. 5.3.1.9); 2,D-mannose-6-phosphate ketol-isomerase (E.C. 5.3.1.8); 3, D-mannose6-phosphate 1,6-phosphomutase (E.C. 5.4.2.8); 4, mannose−1-phosphateguanylyltransferase (E.C. 2.7.7.22); 5, GDP-mannose 3,5-epimerase (E.C.5.1.3.18); 6, galactose−1-phosphate guanylyltransferase (E.C. 2.7.n.n);7, L-galactose 1-phosphate phosphatase (E.C. 3.1.3.n).

FIG. 10 depicts different fermentation pathways from pyruvate toethanol. Each reaction is numbered. Enzymes catalyzing each reaction areas follows: 1, pyruvate decarboxylase (E.C. 4.1.1.1); 2, alcoholdehydrogenase (E.C. 1.1.1.1); 3, pyruvate-formate lyase (E.C. 2.3.1.54);4, acetaldehyde dehydrogenase (E.C. 1.2.1.10); 5, pyruvate synthase(E.C. 1.2.7.1).

FIG. 11 depicts the metabolic reactions of the mevalonate-independentpathway (also known as the non-mevalonate pathway or deoxyxylulose5-phosphate (DXP) pathway) for production of isopentenyl pyrophosphate(IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). In metabolitenames, —P denotes phosphate. Each reaction is numbered. Enzymescatalyzing each reaction are as follows: 1,1-deoxy-D-xylulose-5-phosphate synthase (E.C. 2.2.1.7); 2,1-deoxy-D-xylulose-5-phosphate reductoisomerase (E.C. 1.1.1.267); 3,4-diphosphocytidyl-2C-methyl-D-erythritol synthase (E.C. 2.7.7.60); 4,4-diphosphocytidyl-2C-methyl-D-erythritol kinase (E.C. 2.7.1.148); 5,2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (E.C. 4.6.1.12); 6,(E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (E.C. 1.17.7.1);7, isopentyl/dimethylallyl diphosphate synthase or4-hydroxy-3-methylbut-2-enyl diphosphate reductase (E.C. 1.17.1.2).

FIG. 12 depicts the metabolic reactions of the mevalonate pathway (alsoknown as the HMG-CoA reductase pathway) for production of isopentenylpyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP).In metabolite names, —P denotes phosphate. Each reaction is numbered.Enzymes catalyzing each reaction are as follows: 1, acetyl-CoA thiolase;2, HMG-CoA synthase (E.C. 2.3.3.10); 3, HMG-CoA reductase (E.C.1.1.1.34); 4, mevalonate kinase (E.C. 2.7.1.36); 5, phosphomevalonatekinase (E.C. 2.7.4.2); 6, mevalonate pyrophosphate decarboxylase (E.C.4.1.1.33); 7, isopentenyl pyrophosphate isomerase (E.C. 5.3.3.2).

FIG. 13 depicts the metabolic reactions of the glycerol/1,3-propanediolbiosynthetic pathway for production of glycerol or 1,3-propanediol. Inmetabolite names, —P denotes phosphate. Each reaction is numbered.Enzymes catalyzing each reaction are as follows: 1, sn-glycerol-3-Pdehydrogenase (E.C. 1.1.1.8 or 1.1.1.94); 2, sn-glycerol-3-phosphatase(E.C. 3.1.3.21); 3, sn-glycerol-3-P glycerol dehydratase (E.C.4.2.1.30); 4, 1,3-propanediol oxidoreductase (E.C. 1.1.1.202).

FIG. 14 depicts the metabolic reactions of the polyhydroxybutyratebiosynthetic pathway. Each reaction is numbered. Enzymes catalyzing eachreaction are as follows: 1, acetyl-CoA:acetyl-CoA C-acetyltransferase(E.C. 2.3.1.9); 2, (R)-3-hydroxyacyl-CoA:NADP+oxidoreductase (E.C.1.1.1.36); 3, polyhydroxyalkanoate synthase (E.C. 2.3.1.-).

FIG. 15 depicts the metabolic reactions of one lysine biosynthesispathway. In metabolite names, —P denotes phosphate. Each reaction isnumbered. Enzymes catalyzing each reaction are as follows: 1, aspartateaminotransferase (E.C. 2.6.1.1); 2, aspartate kinase (E.C. 2.7.2.4); 3,aspartate semialdehyde dehydrogenase (E.C. 1.2.1.11); 4,dihydrodipicolinate synthase (E.C. 4.2.1.52); 5, dihydrodipicolinatereductase (E.C. 1.3.1.26); 6, tetrahydrodipicolinate succinylase (E.C.2.3.1.117); 7, N-succinyldiaminopimelate-aminotransferase (E.C.2.6.1.17); 8, N-succinyl-L-diaminopimelate desuccinylase (E.C.3.5.1.18); 9, diaminopimelate epimerase (E.C. 5.1.1.7); 10,diaminopimelate decarboxylase (E.C. 4.1.1.20).

FIG. 16 depicts the metabolic reactions of the γ-valerolactonebiosynthetic pathway. Each reaction is numbered. Enzymes catalyzing eachreaction are as follows: 1, propionyl-CoA synthase (E.C. 6.2.1.-, E.C.4.2.1.- and E.C. 1.3.1.-); 2, beta-ketothiolase (E.C. 2.3.1.16); 3,acetoacetyl-CoA reductase (E.C. 1.1.1.36); 4, 3-hydroxybutyryl-CoAdehydratase (E.C. 4.2.1.55); 5, vinylacetyl-CoA A-isomerase (E.C.5.3.3.3); 6, 4-hydroxybutyryl-CoA transferase (E.C. 2.8.3.-); 7,1,4-lactonase (E.C. 3.1.1.25).

FIG. 17 depicts an example time course of formate usage, formateaccumulation, biomass formation and CO2 emission for a 1-L aerobic,CSTR-type bioreactor initially charged with 0.5 L of minimal mediumcontaining Paracoccus versutus and fed ammonium formate as a sole sourceof carbo and energy at a rate of 10 mM hr⁻¹. Over the course of the runthe working volume changed from 0.5 L to 0.785 L. The data for this runcorresponds to formate consumption rates of 1.6 g L⁻¹ hr⁻¹, maximumbiomass concentrations of 2.5 gDCW L⁻¹, and carbon fixation fluxes of 8mmol-C gDCW⁻¹ L⁻¹.

FIG. 18 depicts the required mass transfer coefficient (K_(L)a) andrequired reactor volume for 0.5 t/d of fuel production, as a function ofmaximum fuel productivity for isooctanol, assuming fuel production fromsynthesis gas for an ideal engineered organism. On the y axis, thetypical range of K_(L)a in large-scale stirred-tank bioreactors isdenoted (A). On the x axis, reported natural formate uptake rates atindustrially relevant culture densities are denoted (B).

DETAILED DESCRIPTION

The present invention relates to developing and using engineered and/orevolved methylotrophs capable of utilizing C1 compounds to produce adesired product. The invention provides for the engineering of amethylotroph, for example, Paracoccus denitrificans, Paracoccus versutusor Paracoccus zeaxanthinifaciens, or other organism suitable forcommercial large-scale production of fuels and chemicals, that canefficiently utilize C1 compounds as a substrate for growth and forchemical production provides cost-advantaged processes for manufacturingof carbon based products of interest. The organisms can be optimized andtested rapidly and at reasonable costs. The invention further providesfor the engineering of a methylotroph to include one or more additionalor alternative pathways for utilization of C1 compounds to producecentral metabolites for growth and/or other desired products.

C1 compounds represent an alternative feedstock to sugar or light pluscarbon dioxide for the production of carbon-based products of interest.There exist non-biological routes to convert C1 compounds to chemicalsand fuels of interest. For example, the Fischer-Tropsch process consumescarbon monoxide and hydrogen gas generated from gasification of coal orbiomass to produce methanol or mixed hydrocarbons as fuels [U.S. Pat.No. 1,746,464]. The drawbacks of Fischer-Tropsch processes are: 1) alack of product selectivity, which results in difficulties separatingdesired products; 2) catalyst sensitivity to poisoning; 3) high energycosts due to high temperatures and pressures required; and 4) thelimited range of products available at commercially competitive costs.Without the advent of carbon sequestration technologies that can operateat scale, the Fischer-Tropsch process is widely considered to be anenvironmentally costly method for generating liquid fuels.Alternatively, processes that rely on naturally occurring microbes thatconvert synthesis gas or syngas, a mixture of primarily molecularhydrogen and carbon monoxide that can be obtained via gasification ofany organic feedstock, such as coal, coal oil, natural gas, biomass, orwaste organic matter, to products such as ethanol, acetate, methane, ormolecular hydrogen are available [Henstra, 2007]. However, thesenaturally occurring microbes can produce only a very restricted set ofproducts, are limited in their efficiencies, lack established tools forgenetic manipulation, and are sensitive to their end products at highconcentrations. Finally, there is some work to introduce syngasutilization into industrial microbial hosts [U.S. Pat. No. 7,803,589];however, these processes have yet to be demonstrated at commercial scaleand are limited to using syngas as the feedstock.

The present invention provides, in some aspects, engineered or evolvedmethylotrophic organisms that are advantageous and/or suitable forindustrial uses. The invention also provides a source of renewableenergy. In some embodiments, the invention provides for the use of a C1compound, such as formate, formic acid, formaldehyde, methanol or anycombination thereof. In one embodiment, the C1 compound can be derivedfrom electrolysis. There is tremendous commercial activity towards thegoal of renewable and/or carbon-neutral energy from solar voltaic,geothermal, wind, nuclear, hydroelectric and more. However, most ofthese technologies produce electricity and are thus limited in use tothe electrical grid [Whipple, 2010]. Furthermore, at least some of theserenewable energy sources such as solar and wind suffer from beingintermittent and unreliable. The lack of practical, large scaleelectricity storage technologies limits how much of the electricitydemand can be shifted to renewable sources. The ability to storeelectrical energy in chemical form, such as in carbon-based products ofinterest, would both offer a means for large-scale electricity storageand allow renewable electricity to meet energy demand from thetransportation sector. Renewable electricity combined with electrolysis,such as the electrochemical production of formate/formic acid fromcarbon dioxide [for example, WO/2007/041872] or formaldehyde or methanolfrom carbon dioxide [for example, WO/2010/088524, WO/2012/015909,WO/2012/015905], opens the possibility of a sustainable, renewablesupply of the C1 compound as one aspect of the present invention.

In some embodiments, the invention provides for the use of a C1compound, such as formaldehyde and/or methanol, derived from wastestreams. For example, formaldehyde is an oxidation product of methanolor methane. Methanol can be prepared from synthesis gas (the majorproduct of gasification of coal, coal oil, natural gas, and ofcarbonaceous materials such as biomass materials, including agriculturalcrops and residues, and waste organic matter) or reductive conversion ofcarbon dioxide and hydrogen by chemical synthetic processes. Methane isa major component of natural gas and can also be obtained from renewablebiomass.

The invention provides for the expression of one or more exogenousproteins or enzymes in the host cell, thereby conferring biosyntheticpathway(s) to utilize central metabolites to produce reduced organiccompounds. The engineered cell can also be endowed with one or morecarbon product biosynthetic pathways that convert central metabolitesinto desired products, such as carbon-based products of interest.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art would understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given thewell-known fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

DEFINITIONS

As used herein, the terms “nucleic acids,” “nucleic acid molecule” and“polynucleotide” may be used interchangeably and include bothsingle-stranded (ss) and double-stranded (ds) RNA, DNA and RNA:DNAhybrids. As used herein the terms “nucleic acid”, “nucleic acidmolecule”, “polynucleotide”, “oligonucleotide”, “oligomer” and “oligo”are used interchangeably and are intended to include, but are notlimited to, a polymeric form of nucleotides that may have variouslengths, including either deoxyribonucleotides or ribonucleotides, oranalogs thereof. For example, oligos may be from 5 to about 200nucleotides, from 10 to about 100 nucleotides, or from 30 to about 50nucleotides long. However, shorter or longer oligonucleotides may beused. Oligos for use in the present invention can be fully designed. Anucleic acid molecule may encode a full-length polypeptide or a fragmentof any length thereof, or may be non-coding.

Nucleic acids can refer to naturally-occurring or synthetic polymericforms of nucleotides. The oligos and nucleic acid molecules of thepresent invention may be formed from naturally-occurring nucleotides,for example forming deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) molecules. Alternatively, the naturally-occurring oligonucleotidesmay include structural modifications to alter their properties, such asin peptide nucleic acids (PNA) or in locked nucleic acids (LNA). Theterms should be understood to include equivalents, analogs of either RNAor DNA made from nucleotide analogs and as applicable to the embodimentbeing described, single-stranded or double-stranded polynucleotides.Nucleotides useful in the invention include, for example,naturally-occurring nucleotides (for example, ribonucleotides ordeoxyribonucleotides), or natural or synthetic modifications ofnucleotides, or artificial bases. Modifications can also includephosphorothioated bases for increased stability.

Nucleic acid sequences that are “complementary” are those that arecapable of base-pairing according to the standard Watson-Crickcomplementarity rules. As used herein, the term “complementarysequences” means nucleic acid sequences that are substantiallycomplementary, as may be assessed by the nucleotide comparison methodsand algorithms set forth below, or as defined as being capable ofhybridizing to the polynucleotides that encode the protein sequences.

As used herein, the term “gene” refers to a nucleic acid that containsinformation necessary for expression of a polypeptide, protein, oruntranslated RNA (e.g., rRNA, tRNA, anti-sense RNA). When the geneencodes a protein, it includes the promoter and the structural gene openreading frame sequence (ORF), as well as other sequences involved inexpression of the protein. When the gene encodes an untranslated RNA, itincludes the promoter and the nucleic acid that encodes the untranslatedRNA.

As used herein, the term “genome” refers to the whole hereditaryinformation of an organism that is encoded in the DNA (or RNA forcertain viral species) including both coding and non-coding sequences.In various embodiments, the term may include the chromosomal DNA of anorganism and/or DNA that is contained in an organelle such as, forexample, the mitochondria or chloroplasts and/or extrachromosomalplasmid and/or artificial chromosome. A “native gene” or “endogenousgene” refers to a gene that is native to the host cell with its ownregulatory sequences whereas an “exogenous gene” or “heterologous gene”refers to any gene that is not a native gene, comprising regulatoryand/or coding sequences that are not native to the host cell. In someembodiments, a heterologous gene may comprise mutated sequences or partof regulatory and/or coding sequences. In some embodiments, theregulatory sequences may be heterologous or homologous to a gene ofinterest. A heterologous regulatory sequence does not function in natureto regulate the same gene(s) it is regulating in the transformed hostcell. “Coding sequence” refers to a DNA sequence coding for a specificamino acid sequence. As used herein, “regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, ribosome binding sites, translation leader sequences, RNAprocessing site, effector (e.g., activator, repressor) binding sites,stem-loop structures, and so on.

As described herein, a genetic element may be any coding or non-codingnucleic acid sequence. In some embodiments, a genetic element is anucleic acid that codes for an amino acid, a peptide or a protein.Genetic elements may be operons, genes, gene fragments, promoters,exons, introns, regulatory sequences, or any combination thereof.Genetic elements can be as short as one or a few codons or may be longerincluding functional components (e.g. encoding proteins) and/orregulatory components. In some embodiments, a genetic element includesan entire open reading frame of a protein, or the entire open readingframe and one or more (or all) regulatory sequences associatedtherewith. One skilled in the art would appreciate that the geneticelements can be viewed as modular genetic elements or genetic modules.For example, a genetic module can comprise a regulatory sequence or apromoter or a coding sequence or any combination thereof. In someembodiments, the genetic element includes at least two different geneticmodules and at least two recombination sites. In eukaryotes, the geneticelement can comprise at least three modules. For example, a geneticmodule can be a regulator sequence or a promoter, a coding sequence, anda polyadenlylation tail or any combination thereof. In addition to thepromoter and the coding sequences, the nucleic acid sequence maycomprises control modules including, but not limited to a leader, asignal sequence and a transcription terminator. The leader sequence is anon-translated region operably linked to the 5′ terminus of the codingnucleic acid sequence. The signal peptide sequence codes for an aminoacid sequence linked to the amino terminus of the polypeptide whichdirects the polypeptide into the cell's secretion pathway.

As generally understood, a codon is a series of three nucleotides(triplets) that encodes a specific amino acid residue in a polypeptidechain or for the termination of translation (stop codons). There are 64different codons (61 codons encoding for amino acids plus 3 stop codons)but only 20 different translated amino acids. The overabundance in thenumber of codons allows many amino acids to be encoded by more than onecodon. Different organisms (and organelles) often show particularpreferences or biases for one of the several codons that encode the sameamino acid. The relative frequency of codon usage thus varies dependingon the organism and organelle. In some instances, when expressing aheterologous gene in a host organism, it is desirable to modify the genesequence so as to adapt to the codons used and codon usage frequency inthe host. In particular, for reliable expression of heterologous genesit may be preferred to use codons that correlate with the host's tRNAlevel, especially the tRNA's that remain charged during starvation. Inaddition, codons having rare cognate tRNA's may affect protein foldingand translation rate, and thus, may also be used. Genes designed inaccordance with codon usage bias and relative tRNA abundance of the hostare often referred to as being “optimized” for codon usage, which hasbeen shown to increase expression level. Optimal codons also help toachieve faster translation rates and high accuracy. In general, codonoptimization involves silent mutations that do not result in a change tothe amino acid sequence of a protein.

Genetic elements or genetic modules may derive from the genome ofnatural organisms or from synthetic polynucleotides or from acombination thereof. In some embodiments, the genetic elements modulesderive from different organisms. Genetic elements or modules useful forthe methods described herein may be obtained from a variety of sourcessuch as, for example, DNA libraries, BAC (bacterial artificialchromosome) libraries, de novo chemical synthesis, or excision andmodification of a genomic segment. The sequences obtained from suchsources may then be modified using standard molecular biology and/orrecombinant DNA technology to produce polynucleotide constructs havingdesired modifications for reintroduction into, or construction of, alarge product nucleic acid, including a modified, partially synthetic orfully synthetic genome. Exemplary methods for modification ofpolynucleotide sequences obtained from a genome or library include, forexample, site directed mutagenesis; PCR mutagenesis; inserting, deletingor swapping portions of a sequence using restriction enzymes optionallyin combination with ligation; in vitro or in vivo homologousrecombination; and site-specific recombination; or various combinationsthereof. In other embodiments, the genetic sequences useful inaccordance with the methods described herein may be syntheticoligonucleotides or polynucleotides. Synthetic oligonucleotides orpolynucleotides may be produced using a variety of methods known in theart.

In some embodiments, genetic elements share less than 99%, less than95%, less than 90%, less than 80%, less than 70% sequence identity witha native or natural nucleic acid sequences. Identity can each bedetermined by comparing a position in each sequence which may be alignedfor purposes of comparison. When an equivalent position in the comparedsequences is occupied by the same base or amino acid, then the moleculesare identical at that position; when the equivalent site occupied by thesame or a similar amino acid residue (e.g., similar in steric and/orelectronic nature), then the molecules can be referred to as homologous(similar) at that position. Expression as a percentage of homology,similarity, or identity refers to a function of the number of identicalor similar amino acids at positions shared by the compared sequences.Expression as a percentage of homology, similarity, or identity refersto a function of the number of identical or similar amino acids atpositions shared by the compared sequences. Various alignment algorithmsand/or programs may be used, including FASTA, BLAST, or ENTREZ FASTA andBLAST are available as a part of the GCG sequence analysis package(University of Wisconsin, Madison, Wis.), and can be used with, e.g.,default settings. ENTREZ is available through the National Center forBiotechnology Information, National Library of Medicine, NationalInstitutes of Health, Bethesda, Md. In one embodiment, the percentidentity of two sequences can be determined by the GCG program with agap weight of 1, e.g., each amino acid gap is weighted as if it were asingle amino acid or nucleotide mismatch between the two sequences.Other techniques for alignment are described [Doolittle, 1996].Preferably, an alignment program that permits gaps in the sequence isutilized to align the sequences. The Smith-Waterman is one type ofalgorithm that permits gaps in sequence alignments [Shpaer, 1997]. Also,the GAP program using the Needleman and Wunsch alignment method can beutilized to align sequences. An alternative search strategy uses MPSRCHsoftware, which runs on a MASPAR computer. MPSRCH uses a Smith-Watermanalgorithm to score sequences on a massively parallel computer.

As used herein, an “ortholog” is a gene or genes that are related byvertical descent and are responsible for substantially the same oridentical functions in different organisms. For example, mouse epoxidehydrolase and human epoxide hydrolase can be considered orthologs forthe biological function of hydrolysis of epoxides. Genes are related byvertical descent when, for example, they share sequence similarity ofsufficient amount to indicate they are homologous, or related byevolution from a common ancestor. Genes can also be considered orthologsif they share three-dimensional structure but not necessarily sequencesimilarity, of a sufficient amount to indicate that they have evolvedfrom a common ancestor to the extent that the primary sequencesimilarity is not identifiable. Genes that are orthologous can encodeproteins with sequence similarity of about 25% to 100% amino acidsequence identity. Genes encoding proteins sharing an amino acidsimilarity less that 25% can also be considered to have arisen byvertical descent if their three-dimensional structure also showssimilarities. Members of the serine protease family of enzymes,including tissue plasminogen activator and elastase, are considered tohave arisen by vertical descent from a common ancestor. Orthologsinclude genes or their encoded gene products that through, for example,evolution, have diverged in structure or overall activity. For example,where one species encodes a gene product exhibiting two functions andwhere such functions have been separated into distinct genes in a secondspecies, the three genes and their corresponding products are consideredto be orthologs. For the production of a biochemical product, thoseskilled in the art would understand that the orthologous gene harboringthe metabolic activity to be introduced or disrupted is to be chosen forconstruction of the non-naturally occurring microorganism. An example oforthologs exhibiting separable activities is where distinct activitieshave been separated into distinct gene products between two or morespecies or within a single species. A specific example is the separationof elastase proteolysis and plasminogen proteolysis, two types of serineprotease activity, into distinct molecules as plasminogen activator andelastase. A second example is the separation of mycoplasma 5′-3′exonuclease and Drosophila DNA polymerase III activity. The DNApolymerase from the first species can be considered an ortholog toeither or both of the exonuclease or the polymerase from the secondspecies and vice versa.

In contrast, as used herein, “paralogs” are homologs related by, forexample, duplication followed by evolutionary divergence and havesimilar or common, but not identical functions. Paralogs can originateor derive from, for example, the same species or from a differentspecies. For example, microsomal epoxide hydrolase (epoxide hydrolase I)and soluble epoxide hydrolase (epoxide hydrolase II) can be consideredparalogs because they represent two distinct enzymes, co-evolved from acommon ancestor, that catalyze distinct reactions and have distinctfunctions in the same species. Paralogs are proteins from the samespecies with significant sequence similarity to each other suggestingthat they are homologous, or related through co-evolution from a commonancestor. Groups of paralogous protein families include HipA homologs,luciferase genes, peptidases, and others.

As used herein, a “nonorthologous gene displacement” is a nonorthologousgene from one species that can substitute for a referenced gene functionin a different species. Substitution includes, for example, being ableto perform substantially the same or a similar function in the speciesof origin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement may beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides can reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences. Exemplary parameters for determining relatedness oftwo or more sequences using the BLAST algorithm, for example, can be asset forth below. Briefly, amino acid sequence alignments can beperformed using BLASTP version 2.0.8 (Jan. 5, 1999) and the followingparameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1;x_dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acidsequence alignments can be performed using BLASTN version 2.0.6 (Sep.16, 1998) and the following parameters: Match: 1; mismatch: −2; gapopen: 5; gap extension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11;filter: off. Those skilled in the art would know what modifications canbe made to the above parameters to either increase or decrease thestringency of the comparison, for example, and determine the relatednessof two or more sequences.

As used herein, the term “homolog” refers to any ortholog, paralog,nonorthologous gene, or similar gene encoding an enzyme catalyzing asimilar or substantially similar metabolic reaction, whether from thesame or different species.

As used herein, the phrase “homologous recombination” refers to theprocess in which nucleic acid molecules with similar nucleotidesequences associate and exchange nucleotide strands. A nucleotidesequence of a first nucleic acid molecule that is effective for engagingin homologous recombination at a predefined position of a second nucleicacid molecule can therefore have a nucleotide sequence that facilitatesthe exchange of nucleotide strands between the first nucleic acidmolecule and a defined position of the second nucleic acid molecule.Thus, the first nucleic acid can generally have a nucleotide sequencethat is sufficiently complementary to a portion of the second nucleicacid molecule to promote nucleotide base pairing. Homologousrecombination requires homologous sequences in the two recombiningpartner nucleic acids but does not require any specific sequences.Homologous recombination can be used to introduce a heterologous nucleicacid and/or mutations into the host genome. Such systems typically relyon sequence flanking the heterologous nucleic acid to be expressed thathas enough homology with a target sequence within the host cell genomethat recombination between the vector nucleic acid and the targetnucleic acid takes place, causing the delivered nucleic acid to beintegrated into the host genome. These systems and the methods necessaryto promote homologous recombination are known to those of skill in theart.

It should be appreciated that the nucleic acid sequence of interest orthe gene of interest may be derived from the genome of naturalorganisms. In some embodiments, genes of interest may be excised fromthe genome of a natural organism or from the host genome, for example E.coli. It has been shown that it is possible to excise large genomicfragments by in vitro enzymatic excision and in vivo excision andamplification. For example, the FLP/FRT site specific recombinationsystem and the Cre/loxP site specific recombination systems have beenefficiently used for excision large genomic fragments for the purpose ofsequencing [Yoon, 1998]. In some embodiments, excision and amplificationtechniques can be used to facilitate artificial genome or chromosomeassembly. Genomic fragments may be excised from the chromosome of amethylotroph and altered before being inserted into the host cellartificial genome or chromosome. In some embodiments, the excisedgenomic fragments can be assembled with engineered promoters and/orother gene expression elements and inserted into the genome of the hostcell.

As used herein, the term “polypeptide” refers to a sequence ofcontiguous amino acids of any length. The terms “peptide,”“oligopeptide,” “protein” or “enzyme” may be used interchangeably hereinwith the term “polypeptide”. In certain instances, “enzyme” refers to aprotein having catalytic activities.

A “proteome” is the entire set of proteins expressed by a genome, cell,tissue or organism. More specifically, it is the set of expressedproteins in a given type of cells or an organism at a given time underdefined conditions. Transcriptome is the set of all RNA molecules,including mRNA, rRNA, tRNA, and other non-coding RNA produced in one ora population of cells. Metabolome refers to the complete set ofsmall-molecule metabolites (such as metabolic intermediates, hormonesand other signaling molecules, and secondary metabolites) to be foundwithin a biological sample, such as a single organism.

The term “fuse,” “fused” or “link” refers to the covalent linkagebetween two polypeptides in a fusion protein. The polypeptides aretypically joined via a peptide bond, either directly to each other orvia an amino acid linker. Optionally, the peptides can be joined vianon-peptide covalent linkages known to those of skill in the art.

As used herein, unless otherwise stated, the term “transcription” refersto the synthesis of RNA from a DNA template; the term “translation”refers to the synthesis of a polypeptide from an mRNA template.Translation in general is regulated by the sequence and structure of the5′ untranslated region (5′-UTR) of the mRNA transcript. One regulatorysequence is the ribosome binding site (RBS), which promotes efficientand accurate translation of mRNA. The prokaryotic RBS is theShine-Dalgarno sequence, a purine-rich sequence of 5′-UTR that iscomplementary to the UCCU core sequence of the 3′-end of 16S rRNA(located within the 30S small ribosomal subunit). Various Shine-Dalgarnosequences have been found in prokaryotic mRNAs and generally lie about10 nucleotides upstream from the AUG start codon. Activity of a RBS canbe influenced by the length and nucleotide composition of the spacerseparating the RBS and the initiator AUG. In eukaryotes, the Kozaksequence A/GCCACCAUGG, which lies within a short 5′ untranslated region,directs translation of mRNA. An mRNA lacking the Kozak consensussequence may also be translated efficiently in an in vitro systems if itpossesses a moderately long 5′-UTR that lacks stable secondarystructure. While E. coli ribosome preferentially recognizes theShine-Dalgarno sequence, eukaryotic ribosomes (such as those found inretic lysate) can efficiently use either the Shine-Dalgarno or the Kozakribosomal binding sites.

As used herein, the terms “promoter,” “promoter element,” or “promotersequence” refer to a DNA sequence which when ligated to a nucleotidesequence of interest is capable of controlling the transcription of thenucleotide sequence of interest into mRNA. A promoter is typically,though not necessarily, located 5′ (i.e., upstream) of a nucleotidesequence of interest whose transcription into mRNA it controls, andprovides a site for specific binding by RNA polymerase and othertranscription factors for initiation of transcription.

One should appreciate that promoters have modular architecture and thatthe modular architecture may be altered. Bacterial promoters typicallyinclude a core promoter element and additional promoter elements. Thecore promoter refers to the minimal portion of the promoter required toinitiate transcription. A core promoter includes a Transcription StartSite, a binding site for RNA polymerases and general transcriptionfactor binding sites. The “transcription start site” refers to the firstnucleotide to be transcribed and is designated +1. Nucleotidesdownstream the start site are numbered +1, +2, etc., and nucleotidesupstream the start site are numbered −1, −2, etc. Additional promoterelements are located 5′ (i.e., typically 30-250 bp upstream of the startsite) of the core promoter and regulate the frequency of thetranscription. The proximal promoter elements and the distal promoterelements constitute specific transcription factor site. In prokaryotes,a core promoter usually includes two consensus sequences, a −10 sequenceor a −35 sequence, which are recognized by sigma factors (see, forexample, [Hawley, 1983]). The −10 sequence (10 bp upstream from thefirst transcribed nucleotide) is typically about 6 nucleotides in lengthand is typically made up of the nucleotides adenosine and thymidine(also known as the Pribnow box). In some embodiments, the nucleotidesequence of the −10 sequence is 5′-TATAAT or may comprise 3 to 6 basespairs of the consensus sequence. The presence of this box is essentialto the start of the transcription. The −35 sequence of a core promoteris typically about 6 nucleotides in length. The nucleotide sequence ofthe −35 sequence is typically made up of the each of the fournucleosides. The presence of this sequence allows a very hightranscription rate. In some embodiments, the nucleotide sequence of the−35 sequence is 5′-TTGACA or may comprise 3 to 6 bases pairs of theconsensus sequence. In some embodiments, the −10 and the −35 sequencesare spaced by about 17 nucleotides. Eukaryotic promoters are morediverse than prokaryotic promoters and may be located several kilobasesupstream of the transcription starting site. Some eukaryotic promoterscontain a TATA box (e.g. containing the consensus sequence TATAAA orpart thereof), which is located typically within 40 to 120 bases of thetranscriptional start site. One or more upstream activation sequences(UAS), which are recognized by specific binding proteins can act asactivators of the transcription. Theses UAS sequences are typicallyfound upstream of the transcription initiation site. The distancebetween the UAS sequences and the TATA box is highly variable and may beup to 1 kb.

As used herein, the term “vector” refers to any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, artificial chromosome,episome, virus, virion, etc., capable of replication when associatedwith the proper control elements and which can transfer gene sequencesinto or between cells. The vector may contain a marker suitable for usein the identification of transformed or transfected cells. For example,markers may provide antibiotic resistant, fluorescent, enzymatic, aswell as other traits. As a second example, markers may complementauxotrophic deficiencies or supply critical nutrients not in the culturemedia. Types of vectors include cloning and expression vectors. As usedherein, the term “cloning vector” refers to a plasmid or phage DNA orother DNA sequence which is able to replicate autonomously in a hostcell and which is characterized by one or a small number of restrictionendonuclease recognition sites and/or sites for site-specificrecombination. A foreign DNA fragment may be spliced into the vector atthese sites in order to bring about the replication and cloning of thefragment. The term “expression vector” refers to a vector which iscapable of expressing of a gene that has been cloned into it. Suchexpression can occur after transformation into a host cell, or in IVPSsystems. The cloned DNA is usually operably linked to one or moreregulatory sequences, such as promoters, activator/repressor bindingsites, terminators, enhancers and the like. The promoter sequences canbe constitutive, inducible and/or repressible.

As used herein, the term “host” or “host cell” refers to any prokaryoticor eukaryotic (e.g., mammalian, insect, yeast, plant, bacterial,archaeal, avian, animal, etc.) cell or organism. The host cell can be arecipient of a replicable expression vector, cloning vector or anyheterologous nucleic acid molecule. In an embodiment, the host cell is amethylotroph (e.g., naturally exsisting or genetically engineered ormetabolically evolved). Host cells may be prokaryotic cells such asspecies of the genus Paracoccus and Escherichia, or eukaryotic cellssuch as yeast, insect, amphibian, or mammalian cells or cell lines. Celllines refer to specific cells that can grow indefinitely given theappropriate medium and conditions. Cell lines can be mammalian celllines, insect cell lines or plant cell lines. Exemplary cell lines caninclude tumor cell lines and stem cell lines. The heterologous nucleicacid molecule may contain, but is not limited to, a sequence ofinterest, a transcriptional regulatory sequence (such as a promoter,enhancer, repressor, and the like) and/or an origin of replication. Asused herein, the terms “host,” “host cell,” “recombinant host” and“recombinant host cell” may be used interchangeably. For examples ofsuch hosts, see [Sambrook, 2001].

One or more nucleic acid sequences can be targeted for delivery totarget prokaryotic or eukaryotic cells via conventional transformationor transfection techniques. As used herein, the terms “transformation”and “transfection” are intended to refer to a variety of art-recognizedtechniques for introducing an exogenous nucleic acid sequence (e.g.,DNA) into a target cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection,conjugation, electroporation, optoporation, injection and the like.Suitable transformation or transfection media include, but are notlimited to, water, CaCl₂, cationic polymers, lipids, and the like.Suitable materials and methods for transforming or transfecting targetcells can be found in [Sambrook, 2001], and other laboratory manuals. Incertain instances, oligo concentrations of about 0.1 to about 0.5micromolar (per oligo) can be used for transformation or transfection.

As used herein, the term “marker” or “reporter” refers to a gene orprotein that can be attached to a regulatory sequence of another gene orprotein of interest, so that upon expression in a host cell or organism,the reporter can confer certain characteristics that can be relativelyeasily selected, identified and/or measured. Reporter genes are oftenused as an indication of whether a certain gene has been introduced intoor expressed in the host cell or organism. Examples of commonly usedreporters include: antibiotic resistance genes, auxotropic markers,β-galactosidase (encoded by the bacterial gene lacZ), luciferase (fromlightning bugs), chloramphenicol acetyltransferase (CAT; from bacteria),GUS (β-glucuronidase; commonly used in plants) and green fluorescentprotein (GFP; from jelly fish). Reporters or markers can be selectableor screenable. A selectable marker (e.g., antibiotic resistance gene,auxotropic marker) is a gene confers a trait suitable for artificialselection; typically host cells expressing the selectable marker isprotected from a selective agent that is toxic or inhibitory to cellgrowth. A screenable marker (e.g., gfp, lacZ) generally allowsresearchers to distinguish between wanted cells (expressing the marker)and unwanted cells (not expressing the marker or expressing atinsufficient level).

As used herein, the term “methylotroph” or “methylotrophic organism”refers to organisms that produce complex organic compounds fromcompounds that lack any carbon-carbon bonds, such as formate, formicacid, formaldehyde, methane, methanol, methylamine, halogenatedmethanes, and methylated sulfur species. Methylotrophs often use C1compounds as both a source of energy and carbon. Example methylotrophicmetabolic pathways for production of central metabolites from C1compounds include the ribulose monophosphate cycle (FIG. 1) and theserine cycle (FIG. 2). “Autotrophs” or “autotrophic organisms” refers toorganisms that use simple, inorganic carbon molecules, such as carbondioxide, as its primary carbon source for growth. Some but not allmethylotrophs assimilate C1 compounds via carbon dioxide and thus arealso autotrophs. These organisms oxidize C1 compounds such as methanol,methylamine, formaldehyde or formate to carbon dioxide (see metabolicpathway depicted in FIG. 3) and then reduce carbon dioxide to centralmetabolites using carbon fixation cycles using, for example, theCalvin-Benson-Bassham cycle (FIG. 4) or the reductive tricarboxlic acidcycle (FIG. 5). In contrast, “heterotrophs” or “heterotrophic organisms”refers to organisms that must use reduced, organic carbon compounds withcarbon-carbon bonds for growth because they cannot use inorganic carbonas their primary carbon source. Instead, heterotrophs obtain energy bybreaking down the organic molecules they consume. Organisms that can usea mix of different sources of energy and carbon are mixotrophs ormixotrophic organisms which can alternate, e.g., between autotrophy andheterotrophy, between autotrophy and methylotrophy, between heterotrophyand methylotrophy, between phototrophy and chemotrophy, betweenlithotrophy and organotrophy, or a combination thereof, depending onenvironmental conditions.

As used herein, the term “reducing cofactor” refers to intracellularredox and energy carriers, such as NADH, NADPH, ubiquinol, menaquinol,cytochromes, flavins and/or ferredoxin, that can donate high energyelectrons in reduction-oxidation reactions. The terms “reducingcofacor”, “reduced cofactor” and “redox cofactor” can be usedinterchangeably.

As used herein, the term “C1 compound”, “1C compound” or “C₁ compound”refers to chemical species that are reduced species but contain nocarbon-carbon bonds. C1 compounds may contain either one carbon atom(e.g., formate, formic acid, formamide, formaldehyde, methane, methanol,methylamine, halogenated methanes, monomethyl sulfate) or multiplecarbon atoms (e.g., dimethyl ether, dimethylamine, dimethyl sulfide).Furthermore, C1 compounds may be either inorganic (e.g., formate, formicacid) or organic e.g., formaldehyde, methane, methanol). C1 compoundsoften serve as both a source of energy and a source of carbon formethylotrophs.

As used herein, the term “central metabolite” refers to organic carboncompounds, such as acetyl-coA, pyruvate, pyruvic acid,3-hydropropionate, 3-hydroxypropionic acid, glycolate, glycolic acid,glyoxylate, glyoxylic acid, dihydroxyacetone phosphate,glyceraldehyde-3-phosphate, malate, malic acid, lactate, lactic acid,acetate, acetic acid, citrate and/or citric acid, that can be convertedinto carbon-based products of interest by a host cell or organism.Central metabolites are generally restricted to those reduced organiccompounds from which all or most cell mass components can be derived ina given host cell or organism. In some embodiments, the centralmetabolite is also the carbon product of interest in which case noadditional chemical conversion is necessary.

Reference to a particular chemical species includes not only thatspecies but also water-solvated forms of the species, unless otherwisestated. For example, carbon dioxide includes not only the gaseous form(CO₂) but also water-solvated forms, such as bicarbonate ion.

As used herein, the term “biosynthetic pathway” or “metabolic pathway”refers to a set of anabolic or catabolic biochemical reactions forconverting (transmuting) one chemical species into another. Anabolicpathways involve constructing a larger molecule from smaller molecules,a process requiring energy. Catabolic pathways involve breaking down oflarger molecules, often releasing energy. As used herein, the term“energy conversion pathway” refers to a metabolic pathway that transfersenergy from a C1 compound to a reducing cofactor. The term “carbonfixation pathway” refers to a biosynthetic pathway that convertsinorganic carbon, such as carbon dioxide, bicarbonate or formate, toreduced organic carbon, such as one or more carbon product precursors.The term “methylotrophic pathway” refers to a biosynthetic pathway thatconverts C1 compounds to compounds with carbon-carbon bonds, such as oneor more carbon product precursors. The term “carbon product biosyntheticpathway” refers to a biosynthetic pathway that converts one or morecarbon product precursors to one or more carbon based products ofinterest.

As used herein, the term “engineered methylotroph” or “engineeredmethylotrophic organism” refers to organisms that have been geneticallyengineered to convert C1 compounds, such as formate, formic acid,formaldehyde, or methanol, to organic carbon compounds. As used herein,an engineered methylotroph need not derive its organic carbon compoundssolely from C1 compounds. The term engineered methylotroph may also beused to refer to originally methylotrophic or mixotrophic organisms thathave been genetically engineered to include one or more energyconversion, carbon fixation, methylotrophic and/or carbon productbiosynthetic pathways in addition or instead of its endogenousmethylotrophic capability. The term “engineer,” “engineering” or“engineered,” as used herein, refers to genetic manipulation ormodification of biomolecules such as DNA, RNA and/or protein, or liketechnique commonly known in the biotechnology art.

As used herein, the term “carbon based products of interest” refers to adesired product containing carbon atoms and include, but not limited toalcohols such as ethanol, propanol, isopropanol, butanol, octanol, fattyalcohols, fatty acid esters, wax esters; hydrocarbons and alkanes suchas propane, octane, diesel, Jet Propellant 8, polymers such asterephthalate, 1,3-propanediol, 1,4-butanediol, polyols,polyhydroxyalkanoates (PHAs), polyhydroxybutyrates (PHBs), acrylate,adipic acid, epsilon-caprolactone, isoprene, caprolactam, rubber;commodity chemicals such as lactate, docosahexaenoic acid (DHA),3-hydroxypropionate, γ-valerolactone, lysine, serine, aspartate,aspartic acid, sorbitol, ascorbate, ascorbic acid, isopentenol,lanosterol, omega-3 DHA, lycopene, itaconate, 1,3-butadiene, ethylene,propylene, succinate, citrate, citric acid, glutamate, malate,3-hydroxyprionic acid (HPA), lactic acid, THF, gamma butyrolactone,pyrrolidones, hydroxybutyrate, glutamic acid, levulinic acid, acrylicacid, malonic acid; specialty chemicals such as carotenoids,isoprenoids, itaconic acid; biological sugars such as glucose, fructose,lactose, sucrose, starch, cellulose, hemicellulose, glycogen, xylose,dextrose, galactose, uronic acid, maltose, polyketides, or glycerol;central metabolites, such as acetyl-coA, pyruvate, pyruvic acid,3-hydropropionate, 3-hydroxypropionic acid, glycolate, glycolic acid,glyoxylate, glyoxylic acid, dihydroxyacetone phosphate,glyceraldehyde-3-phosphate, malate, malic acid, lactate, lactic acid,acetate, acetic acid, citrate and/or citric acid, from which othercarbon products can be made; pharmaceuticals and pharmaceuticalintermediates such as 7-aminodesacetoxycephalosporonic acid,cephalosporin, erythromycin, polyketides, statins, paclitaxel,docetaxel, terpenes, peptides, steroids, omega fatty acids and othersuch suitable products of interest. Such products are useful in thecontext of biofuels, industrial and specialty chemicals, asintermediates used to make additional products, such as nutritionalsupplements, neutraceuticals, polymers, paraffin replacements, personalcare products and pharmaceuticals.

As used herein, the term “hydrocarbon” referes a chemical compound thatconsists of the elements carbon, hydrogen and optionally, oxygen.“Surfactants” are substances capable of reducing the surface tension ofa liquid in which they are dissolved. They are typically composed of awater-soluble head and a hydrocarbon chain or tail. The water solublegroup is hydrophilic and can either be ionic or nonionic, and thehydrocarbon chain is hydrophobic. The term “biofuel” is any fuel thatderives from a biological source.

The accession numbers provided throughout this description are derivedfrom the NCBI database (National Ceter for Biotechnology Information)maintained by the National Institute of Health, USA. The accessionnumbers are provided in the database on Aug. 1, 2011. The EnzymeClassification Numbers (E.C.) provided throughout this description arederived from the KEGG Ligand database, maintained by the KyotoEncyclopedia of Genes and Genomics, sponsored in part by the Universityof Tokyo. The E.C. numbers are provided in the database on Aug. 1, 2011.

Other terms used in the fields of recombinant nucleic acid technology,microbiology, metabolic engineering, and molecular and cell biology asused herein will be generally understood by one of ordinary skill in theapplicable arts.

Source of C1 Compounds

In some embodiments, suitable C1 compounds include, but not limited toformate, formic acid, methanol and/or formaldehyde. Formate, formicacid, formaldehyde and methanol can be produced via the electrochemicalreduction of CO₂ [see, e.g., Hori, 2008].

In some instances, soluble, liquid feedstocks such as formate, formicacid, formaldehyde or methanol can be preferable to gaseous feedstocks,such as methane or synthesis gas. Methane is generally known as a gaswith low water solubility in water which creates mass transferlimitations when using methane as the feedstock for engineered and/orevolved methylotrophs (biological systems are aqueous). Similarly,synthesis gas (composed of molecular hydrogen and carbon monoxide) alsohas low water solubility in water. At large reactor or fermentor scales,high rates of mass transfer from the gas to liquid phases ischallenging. In contrast, formate, formaldehyde and methanol due totheir higher solubility/miscibility in H₂O, do not have this problem.Hence, when water is the solvent in the growth media, the use offormate, formic acid, formaldehyde or methanol as the feedstock can bemore advantageous.

The energy efficiency of electrochemical conversion of carbon dioxideimpacts the overall energy efficiency of a bio-manufacturing processusing an engineered and/or evolved methylotroph of the presentinvention. Electrolyzers achieve overall energy efficiencies of 56-73%at current densities of 110-300 mA/cm² (alkaline electrolyzers) or800-1600 mA/cm² (PEM electrolyzers) [Whipple, 2010]. In contrast,electrochemical systems to date have achieved moderate energyefficiencies or high current densities but not at the same time. Hence,additional technology improvements are needed for electrochemicalproduction of formate, formic acid, formaldehyde and methanol.

Organisms or Host Cells for Engineering or Evolution

The host cell or organism, as disclosed herein, may be chosen frommethylotrophic eukaryotic or prokaryotic systems, such as bacterialcells (Gram-negative (e.g., Alphaproteobacterium) or Gram-positive),archaea and yeast cells. Suitable cells and cell lines can also includethose commonly used in laboratories and/or industrial applications. Insome embodiments, host cells/organisms can be selected from Bacillusspecies including Bacillus methanolicus, Bilophila wadsworthia,Burkholderia species including Burkholderia phymatum, Candida speciesincluding Candida boidinii, Candida sonorensis, Cupravidus necator(formerly Alcaligenes eutrophus and Ralstonia eutropha), Hyphomicrobiumspecies including Hyphomicrobium methylovorum, Hyphomicrobiumzavarzinii, Illethanococcus maripaludis, Methanomonas methanooxidans,Methanosarcina species, Methylibium petroleiphilum, Methylobacillusflagellatus, Methylobacillus flagellatum, Methylobacillusfructoseoxidans, Methylobacillus glycogenes, Methylobacillus viscogenes,Methylobacter bovis, Methylobacter capsulatus, Methylobacter vinelandii,Methylobacterium species including Methylobacterium dichloromethanicum,Methylobacterium extorquens, Methylobacterium mesophilicum,Methylobacterium organophilum, Methylobacterium rhodesianum,Methylococcus capsulatus, Methylococcus minimus, Methylocystis speciesincluding Methylocystis parvus, Methylomicrobium alcaliphilum,Methylomonas species including Methylomonas agile, Methylomonas albus,Methylomonas clara, Methylomonas methanica (formerly Bacillus methanicusand Pseudomonas methanica), Methylomonas methanolica, Methylomonasrosaceous, Methylomonas rubrum, Methylomonas streptobacterium,Methylophilus methylotrophus, Methylosinus species includingMethylosinus sporium, Methylosinus trichosporium, Methylosporovibriomethanica, Methyloversatilis universalis, Methylovorus mays,Mycobacterium vaccae, Nautilia sp. strain AmN, Nautilia lithotrophica,Nautilia profundicola, Paracoccus species including Paracoccusdenitrificans, Paracoccus versutus or Paracoccus zeaxanthinifaciens,Picchia species including Picchia angusta (formerly Hansenulapolymorpha), Picchia guilliermondii, Picchia pastoris, Protaminobacterruber, Pseudomonas species including Pseudomonas AM1, Pseudomonasmethanitrificans, Schlegelia plantiphila, Thermocrinus ruber,Verrucomicrobia species, Xanthobacter species, or any modificationsand/or derivatives thereof. Those skilled in the art would understandthat the genetic modifications, including metabolic alterationsexemplified herein, are described with reference to a suitable hostorganism such as Paracoccus denitrificans and their correspondingmetabolic reactions or a suitable source organism for desired nucleicacids such as genes for a desired metabolic pathway. However, given thecomplete genome sequencing of a wide variety of organisms and the highlevel of skill in the area of genomics, those skilled in the art wouldreadily be able to apply the teachings and guidance provided herein toessentially all other methylotrophic host cells and organisms. Forexample, the Paracoccus denitrificans metabolic modificationsexemplified herein can readily be applied to other species byincorporating the same or analogous encoding nucleic acid from speciesother than the referenced species. Such genetic modifications include,for example, genetic alterations of species homologs, in general, and inparticular, orthologs, paralogs or nonorthologous gene displacements.

In various aspects of the invention, the cells are geneticallyengineered and/or metabolically evolved, for example, for the purposesof optimized energy conversion, methylotrophy and/or carbon fixation.The terms “metabolically evolved” or “metabolic evolution” relates togrowth-based selection (metabolic evolution) of host cells thatdemonstrate improved growth (cell yield).

Exemplary genomes and nucleic acids include full and partial genomes ofa number of organisms for which genome sequences are publicly availableand can be used with the disclosed methods, such as, but not limited to,Aeropyrum pernix; Agrobacterium tumefaciens; Anabaena; Anophelesgambiae; Apis mellifera; Aquifex aeolicus; Arabidopsis thaliana;Archaeoglobus fulgidus; Ashbya gossypii; Bacillus anthracis; Bacilluscereus; Bacillus halodurans; Bacillus licheniformis; Bacillus subtilis;Bacteroides fragilis; Bacteroides thetaiotaomicron; Bartonella henselae;Bartonella quintana; Bdellovibrio bacteriovirus; Bifidobacterium longum;Blochmannia floridanus; Bordetella bronchiseptica; Bordetellaparapertussis; Bordetella pertussis; Borrelia burgdorferi;Bradyrhizobium japonicum; Brucella melitensis; Brucella suis; Buchneraaphidicola; Burkholderia mallei; Burkholderia pseudomallei;Caenorhabditis briggsae; Caenorhabditis elegans; Campylobacter jejuni;Candida glabrata; Canis familiaris; Caulobacter crescentus; Chlamydiamuridarum; Chlamydia trachomatis; Chlamydophila caviae; Chlamydophilapneumoniae; Chlorobium tepidum; Chromobacterium violaceum; Cionaintestinalis; Clostridium acetobutylicum; Clostridium perfringens;Clostridium tetania Corynebacterium diphtheriae; Corynebacteriumefficiens; Coxiella burnetii; Cryptosporidium hominis; Cryptosporidiumparvum; Cyanidioschyzon merolae; Debaryomyces hansenii; Deinococcusradiodurans; Desulfotalea psychrophile; Desulfovibrio vulgaris;Drosophila melanogaster; Encephalitozoon cuniculi; Enterococcusfaecalis; Erwinia carotovora; Escherichia coli; Fusobacteriumnucleaturn; Gallus gallus; Geobacter sulfurreducens; Gloeobacterviolaceus; Guillardia theta; Haemophilus ducreyi; Haemophilusinfluenzae; Halobacterium; Helicobacter hepaticus; Helicobacter pylori;Homo sapiens; Kluyveromyces waltii; Lactobacillus johnsonii;Lactobacillus plantarum; Legionella pneumophila; Leifsonia xyli;Lactococcus lactis; Leptospira interrogans; Listeria innocua; Listeriamonocytogenes; Magnaporthe grisea; Mannheimia succiniciproducens;Mesoplasma florum; Mesorhizobium loti; Methanobacteriumthermoautotrophicum; Methanococcoides burtonii; Methanococcusjannaschii; Methanococcus maripaludis; Methanogenium frigidum;Methanopyrus kandleri; Methanosarcina acetivorans; Methanosarcina mazei;Methylococcus capsulatus; Mus musculus; Mycobacterium bovis;Mycobacterium leprae; Mycobacterium paratuberculosis; Mycobacteriumtuberculosis; Mycoplasma gallisepticum; Mycoplasma genitalium;Mycoplasma mycoides; Mycoplasma penetrans; Mycoplasma pneumoniae;Mycoplasma pulmonis; Mycoplasma mobile; Nanoarchaeum equitans; Neisseriameningitidis; Neurospora crassa; Nitrosomonas europaea; Nocardiafarcinica; Oceanobacillus iheyensis; Onions yellows phytoplasma; Oryzasativa; Pan troglodytes; Paracoccus denitrificans; Paracoccus versutus;Paracoccus zeaxanthinifaciens; Pasteurella multocida; Phanerochaetechrysosporium; Photorhabdus luminescens; Picrophilus torridus;Plasmodium falciparum; Plasmodium yoelii yoelii; Populus trichocarpa;Porphyromonas gingivalis Prochlorococcus marinus; Propionibacteriumacnes; Protochlamydia amoebophila; Pseudomonas aeruginosa; Pseudomonasputida; Pseudomonas syringae; Pyrobaculum aerophilum; Pyrococcus abyssi;Pyrococcus furiosus; Pyrococcus horikoshii; Pyrolobus fumarii; Ralstoniasolanacearum; Rattus norvegicus; Rhodopirellula baltica;Rhodopseudomonas palustris; Rickettsia conorii; Rickettsia typhi;Rickettsia prowazekii; Rickettsia sibirica; Saccharomyces cerevisiae;Saccharomyces bayanus; Saccharomyces boulardii; Saccharopolysporaerythraea; Schizosaccharomyces pombe; Salmonella enterica; Salmonellatyphimurium; Schizosaccharomyces pornbe; Shewanella oneidensis; Shigellaflexneria; Sinorhizobium meliloti; Staphylococcus aureus; Staphylococcusepidermidis; Streptococcus agalactiae; Streptococcus mutans;Streptococcus pneumoniae; Streptococcus pyogenes; Streptococcusthermophilus; Streptomyces avermitilis; Streptomyces coelicolor;Sulfolobus solfataricus; Sulfolobus tokodaii; Synechococcus;Synechoccous elongates; Synechocystis; Takifugu rubripes; Tetraodonnigroviridis; Thalassiosira pseudonana; Thermoanaerobactertengcongensis; Thermoplasma acidophilum; Thermoplasma volcanium;Thermosynechococcus elongatus; Thermotagoa maritima; Thermusthermophilus; Treponema denticola; Treponema pallidum; Tropherymawhipplei; Ureaplasma urealyticum; Vibrio cholerae; Vibrioparahaemolyticus; Vibrio vulnificus; Wigglesworthia glossinidia;Wolbachia pipientis; Wolinella succinogenes; Xanthomonas axonopodis;Xanthomonas campestris; Xylella fastidiosa; Yarrowia lipolytica;Yersinia pseudotuberculosis; and Yersinia pestis nucleic acids.

In certain embodiments, sources of encoding nucleic acids for enzymesfor a biosynthetic pathway can include, for example, any species wherethe encoded gene product is capable of catalyzing the referencedreaction. Exemplary species for such sources include, for example,Aeropyrum pernix; Aquifex aeolicus; Aquifex pyrophilus; CandidatusArcobacter sulfidicus; Candidatus Endoriftia persephone; CandidatusNitrospira defluvii; Chlorobium limicola; Chlorobium tepidum;Clostridium pasteurianum; Desulfobacter hydrogenophilus;Desulfurobacterium thermolithotrophum; Geobacter metallireducens;Halobacterium sp. NRC-1; Hydrogenimonas thermophila; Hydrogenivirgastrain 128-5-R1; Hydrogenobacter thermophilus; Hydrogenobaculum sp.Y04AAS1; Lebetimonas acidiphila Pd55^(T) ; Leptospirillum ferriphilum;Leptospirillum ferrodiazotrophum; Leptospirillum rubarum; Magnetococcusmarinus; Magnetospirillum magneticum; Mycobacterium bovis; Mycobacteriumtuberculosis; Methylobacterium nodulans; Nautilia lithotrophica;Nautilia profundicola; Nautilia sp. strain AmN; Nitratifractorsalsuginis; Nitratiruptor sp. strain SB155-2; Paracoccus denitrificans;Paracoccus versutus; Paracoccus zeaxanthinifaciens; Persephonellamarina; Rimcaris exoculata episymbiont; Streptomyces avermitilis;Streptomyces coelicolor; Sulfolobus avermitilis; Sulfolobussolfataricus; Sulfolobus tokodaii; Sulfurihydrogenibium azorense;Sulfurihydrogenibium sp. Y03AOP1; Sulfurihydrogenibium yellowstonense;Sulfurihydrogenibium subterraneum; Sulfurimonas autotrophica;Sulfurimonas denitrificans; Sulfurimonas paralvinella; Sulfurovumlithotrophicum; Sulfurovum sp. strain NBC37-1; Thermocrinis ruber;Thermovibrio ammonificans; Thermovibrio ruber; Thioreductor micatisoli;Nostoc sp. PCC 7120; Acidithiobacillus ferrooxidans; Allochromatiumvinosum; Aphanothece halophytica; Oscillatoria limnetica; Rhodobactercapsulatus; Thiobacillus denitrificans; Cupriavidus necator (formerlyRalstonia eutropha), Methanosarcina barkeri; Methanosarcia mazei;Methanococcus maripaludis; Mycobacterium smegmatis; Burkholderiastabilis; Candida boidinii; Candida methylica; Pseudomonas sp. 101;Methylcoccus capsulatus; Mycobacterium gastri; Cenarchaeum symbiosum;Chloroflexus aurantiacus; Erythrobacter sp. NAP1; Metallosphaera sedula;gamma proteobacterium NOR51-B; marine gamma proteobacterium HTCC2080;Nitrosopumilus maritimus; Roseiflexus castenholzii; Synechococcuselongatus; and the like, as well as other exemplary species disclosedherein or available as source organisms for corresponding genes.However, with the complete genome sequence publicly available for nowmore than 4400 species (including viruses), including 1701 microbialgenomes and a variety of yeast, fungi, plant, and mammalian genomes, theidentification of genes encoding the requisite energy conversion,methylotrophic, carbon fixation or carbon product biosynthetic activityfor one or more genes in related or distant species, including forexample, homologs, orthologs, paralogs and nonorthologous genedisplacements of known genes, and the replacement of gene homolog eitherwithin a particular engineered and/or evolved methylotroph or betweendifferent host cells for the engineered and/or evolved methylotroph isroutine and well known in the art. Accordingly, the metabolicmodifications enabling methylotrophic growth and production ofcarbon-based products described herein with reference to a particularorganism such as Paracoccus denitrificans can be readily applied toother methylotrophic microorganisms, including prokaryotic andeukaryotic organisms alike. Given the teachings and guidance providedherein, those skilled in the art would know that a metabolicmodification exemplified in one organism can be applied equally to otherorganisms.

In some instances, such as when an alternative energy conversion, carbonfixation, methylotrophic or carbon product biosynthetic pathway existsin an unrelated species, enhanced methylotrophic growth and productionof carbon-based products can be conferred onto the host species by, forexample, exogenous expression of a paralog or paralogs from theunrelated species that catalyzes a similar, yet non-identical metabolicreaction to replace the referenced reaction. Because certain differencesamong metabolic networks exist between different organisms, thoseskilled in the art would understand that the actual gene usage betweendifferent organisms may differ. However, given the teachings andguidance provided herein, those skilled in the art also would understandthat the teachings and methods of the invention can be applied to allmicrobial organisms using the cognate metabolic modifications to thoseexemplified herein to construct a microbial organism in a species ofinterest that would produce carbon-based products of interest from C1compounds.

It should be noted that various engineered strains and/or mutations ofthe organisms or cell lines discussed herein can also be used.

Methods for Identification and Selection of Candidate Enzymes for aMetabolic Activity of Interest

In one aspect, the present invention provides a method for identifyingcandidate proteins or enzymes of interest capable of performing adesired metabolic activity. Leveraging the exponential growth of geneand genome sequence databases and the availability of commercial genesynthesis at reasonable cost, Bayer and colleagues adopted a syntheticmetagenomics approach to bioinformatically search sequence databases forhomologous or similar enzymes, computationally optimize their encodinggene sequences for heterologous expression, synthesize the designed genesequence, clone the synthetic gene into an expression vector and screenthe resulting enzyme for a desired function in E. coli or yeast [Bayer,2009]. However, depending on the metabolic activity or protein ofinterest, there can be thousands of putative homologs in the publiclyavailable sequence databases. Thus, it can be experimentally challengingor in some cases infeasible to synthesize and screen all possiblehomologs at reasonable cost and within a reasonable timeframe. Toaddress this challenge, in one aspect, this invention provides analternate method for identifying and selecting candidate proteinsequences for a metabolic activity of interest. The method comprises thefollowing steps. First, for a desired metabolic activity, such as anenzyme-catalyzed step in an energy conversion, methylotrophic, carbonfixation or carbon product biosynthetic pathway, one or more enzymes ofinterest are identified. Typically, the enzyme(s) of interest have beenpreviously experimentally validated to perform the desired activity, forexample in the published scientific literature. In some embodiments, oneor more of the enzymes of interest has been heterologously expressed andexperimentally demonstrated to be functional. Second, a bioinformaticsearch is performed on protein classification or grouping databases,such as Clusters of Orthologous Groups (COGs) [Tatusov, 1997; Tatusov,2003], Entrez Protein Clusters (ProtClustDB) [Klimke, 2009] and/orInterPro [Zdobnov, 2001], to identify protein groupings that contain oneor more of the enzyme(s) of interest (or closely related enzymes). Ifthe enzyme(s) of interest contain multiple subunits, then the proteincorresponding to a single subunit, for example the catalytic subunit orthe largest subunit, is selected as being representative of theenzyme(s) of interest for the purposes of bioinformatic analysis. Third,a systematic, expert-guided search is then performed to identify whichdatabase groupings are likely to contain a majority of members whosemetabolic activity is the same or similar as the protein(s) of interest.Fourth, the list of NCBI Protein accession numbers corresponding toevery members of each selected database grouping is then compiled andthe corresponding protein sequences are downloaded from the sequencedatabases. Protein sequences available from sources other than thepublic sequence databases may be added to this set. Fifth, optionally,one or more outgroup protein sequences are identified and added to theset. Outgroup proteins are proteins which may share some functional,structural, or sequence similarities to the model enzyme(s) but lack anessential feature of the enzyme(s) of interest or desired metabolicactivity. For example, the enzyme flavocytochrome c (E.C. 1.8.2.3) issimilar to sulfide-quinone oxidoreductase (E.C. 1.8.5.4) in that itoxidizes hydrogen sulfide but it reduces cytochrome c instead ofubiquinone and thus offers a useful outgroup during bioinformaticanalysis of sulfide-quinone oxidoreductases. Sixth, the complete set ofprotein sequences are aligned with an sequence alignment program capableof aligning large numbers of sequences, such as MUSCLE [Edgar, 2004a;Edgar, 2004b]. Seventh, a tree is drawn based on the resulting MUSCLEalignment via methods known to those skilled in the art, such asneighbor joining [Saitou, 1987] or UPGMA [Sokal, 1958; Murtagh, 1984].Eighth, different clades are selected from the tree so that the numberof clades equals the desired number of proteins for screening. Finally,one protein from each Glade is selected for gene synthesis andfunctional screening based on the following heuristics

-   -   Preference is given to proteins that have been heterologously        expressed and experimentally demonstrated to have the desired        metabolic activity.    -   Preference is given to proteins that have been biochemically        characterized to have the desired metabolic activity previously.    -   Preference is given to proteins from source organisms for which        there is strong experimental or genomic evidence that the        organism has the desired metabolic activity.    -   Preference is given to proteins in which the key catalytic,        binding and/or other signature residues are conserved with        respect to the protein(s) of interest.    -   Preference is given to protein from source organisms whose        optimal growth temperature is similar to that of the host cell        or organism. For example, if the host cell is a mesophile, then        the source organism is also a mesophile.

Therefore, in constructing the engineered and/or evolved methylotroph ofthe invention, those skilled in the art would understand that byapplying the teaching and guidance provided herein, it is possible toreplace or augment particular genes within a metabolic pathway, such asan energy conversion pathway, a carbon fixation pathway, amethylotrophic pathway and/or a carbon product biosynthetic pathway,with homologs identified using the methods described here, whose geneproducts catalyze a similar or substantially similar metabolic reaction.Such modifications can be done, for example, to increase flux through ametabolic pathway (for example, flux of energy or carbon), to reduceaccumulation of toxic intermediates, to improve the kinetic propertiesof the pathway, and/or to otherwise optimize the engineered and/orevolved methylotroph.

Methods for Design of Nucleic Acids Encoding Enzymes for HeterologousExpression

In one aspect, the present invention provides a computer program productfor designing a nucleic acid that encodes a protein or enzyme ofinterest that is codon optimized for the host cell or organism (thetarget species). The program can reside on a hardware computer readablestorage medium and having a plurality of instructions which, whenexecuted by a processor, cause the processor to perform operations. Theprogram comprises the following operations. At each amino acid positionof the protein of interest, the codon is selected in which the rankorder codon usage frequency of that codon in the target species is thesame as the rank order codon usage frequency of the codon that occurs atthat position in the source species gene. To select the desired codon ateach amino acid position, both the genetic code (the mapping of codonsto amino acids [Jukes, 1993]) and codon frequency table (the frequencywith which each synonymous codon occurs in a genome or genome [Grantham,1980]) for both the source and target species are needed. For sourcespecies for which a complete genome sequence is available, the usagefrequency for each codon may be calculate simply by summing the numberof instances of that codon in all annotated coding sequences, dividingby the total number of codons in that genome, and then multiplying by1000. For source species for which no complete genome is available, theusage frequency can be computed based on any available coding sequencesor by using the codon frequency table of a closely related organism. Theprogram then preferably standardizes the start codon to ATG, the stopcodon to TAA, and the second and second last codons to one of twentypossible codons (one per amino acid). The program then subjects thecodon optimized nucleic acid sequence to a series of checks to improvethe likelihood that the sequence can be synthesized via commercial genesynthesis and subsequently manipulated via molecular biology [Sambrook,2001] and DNA assembly methods [WO/2010/070295]. These checks compriseidentifying if key restriction enzyme recognition sites used in a DNAassembly standard or DNA assembly method are present; if hairpins whoseGC content exceeds a threshold percentage, such as 60%, and whose lengthexceeds a threshold number of base pairs, such as 10, are present; ifsequence repeats are present; if any subsequence between 100 and 150nucleotides in length exceeds a threshold GC content, such as 65%; if Gor C homopolymers greater than 5 nucleotides in length are present; and,optionally, if any sequence motifs are present that might give rise tospurious transposon insertion sites, transcriptional or translationalinitiation or termination, mRNA secondary structure, RNase cleavage,and/or transcription factor binding. If the codon optimized nucleic acidsequence fails any of these checks, the program then iterates throughall possible synonymous mutations and designs a new nucleic acidsequence that both passes all checks and minimizes the difference incodon frequencies between the original and new nucleic acid sequence.

Various implementations of the systems and techniques described here canbe realized in digital electronic circuitry, integrated circuitry,specially designed ASICs (application-specific integrated circuits),computer hardware, firmware, software, and/or combinations thereof.These various implementations can include one or more computer programsthat are executable and/or interpretable on a programmable systemincluding at least one programmable processor, which may be special orgeneral purpose, coupled to receive data and instructions from, and totransmit data and instructions to, a storage system, at least one inputdevice, and at least one output device. Such computer programs (alsoknown as programs, software, software applications or code) may includemachine instructions for a programmable processor, and may beimplemented in any form of programming language, including high-levelprocedural and/or object-oriented programming languages, and/or inassembly/machine languages. A computer program may be deployed in anyform, including as a stand-alone program, or as a module, component,subroutine, or other unit suitable for use in a computing environment. Acomputer program may be deployed to be executed or interpreted on onecomputer or on multiple computers at one site, or distributed acrossmultiple sites and interconnected by a communication network.

A computer program may, in an embodiment, be stored on a computerreadable storage medium. A computer readable storage medium storescomputer data, which data can include computer program code that isexecuted and/or interpreted by a computer system or processor. By way ofexample, and not limitation, a computer readable medium may comprisecomputer readable storage media, for tangible or fixed storage of data,or communication media for transient interpretation of code-containingsignals. Computer readable storage media, may refer to physical ortangible storage (as opposed to signals) and may include withoutlimitation volatile and non-volatile, removable and non-removable mediaimplemented in any method or technology for the tangible storage ofinformation such as computer-readable instructions, data structures,program modules or other data. Computer readable storage media includes,but is not limited to, RAM, ROM, EPROM, EEPROM, flash memory or othersolid state memory technology, CD-ROM, DVD, or other optical storage,magnetic cassettes, magnetic tape, magnetic disk storage or othermagnetic storage devices, or any other physical or material medium whichcan be used to tangibly store the desired information or data orinstructions and which can be accessed by a computer or processor.

FIG. 6 shows a block diagram of a generic processing architecture, whichmay execute software applications and processes. Computer processingdevice 200 may be coupled to display 202 for graphical output. Processor204 may be a computer processor capable of executing software. Typicalexamples of processor 204 are general-purpose computer processors (suchas Intel® or AMD® processors), ASICs, microprocessors, any other type ofprocessor, or the like. Processor 204 may be coupled to memory 206,which may be a volatile memory (e.g. RAM) storage medium for storinginstructions and/or data while processor 204 executes. Processor 204 mayalso be coupled to storage device 208, which may be a non-volatilestorage medium such as a hard drive, FLASH drive, tape drive, DVDROM, orsimilar device. Program 210 may be a computer program containinginstructions and/or data, and may be stored on storage device 208 and/orin memory 206, for example. In a typical scenario, processor 204 mayload some or all of the instructions and/or data of program 210 intomemory 206 for execution.

Program 210 may be a computer program capable of performing theprocesses and functions described above. Program 210 may include variousinstructions and subroutines, which, when loaded into memory 206 andexecuted by processor 204 cause processor 204 to perform variousoperations, some or all of which may effectuate the methods, processes,and/or functions associated with the presently disclosed embodiments.

Although not shown, computer processing device 200 may include variousforms of input and output. The I/O may include network adapters, USBadapters, Bluetooth radios, mice, keyboards, touchpads, displays, touchscreens, LEDs, vibration devices, speakers, microphones, sensors, or anyother input or output device for use with computer processing device200.

Methods for Expression of Heterologous Enzymes

Composite nucleic acids can be constructed to include one or more energyconversion, methylotrophic, carbon fixation and/or carbon productbiosynthetic pathway encoding nucleic acids as exemplified herein. Thecomposite nucleic acids can subsequently be transformed or transfectedinto a suitable host organism for expression of one or more proteins ofinterest. Composite nucleic acids can be constructed by operably linkingnucleic acids encoding one or more standardized genetic parts withprotein(s) of interest encoding nucleic acids that have also beenstandardized. Standardized genetic parts are nucleic acid sequences thathave been refined to conform to one or more defined technical standards,such as an assembly standard [Knight, 2003; Shetty, 2008; Shetty, 2011].Standardized genetic parts can encode transcriptional initiationelements, transcriptional termination elements, translational initiationelements, translational termination elements, protein affinity tags,protein degradation tags, protein localization tags, selectable markers,replication elements, recombination sites for integration onto thegenome, and more. Standardized genetic parts have the advantage thattheir function can be independently validated and characterized [Kelly,2009] and then readily combined with other standardized parts to producefunctional nucleic acids [Canton, 2008]. By mixing and matchingstandardized genetic parts encoding different expression controlelements with nucleic acids encoding proteins of interest, transformingthe resulting nucleic acid into a suitable host cell and functionallyscreening the resulting engineered cell, the process of both achievingsoluble expression of proteins of interest and validing the function ofthose proteins is made dramatically faster. For example, the set ofstandardized parts might comprise constitutive promoters of varyingstrengths [Davis, 2011], ribosome binding sites of varying strengths[Anderson, 2007] and protein degradation of tags of varying strengths[Andersen, 1998].

For exogenous expression in Paracoccus or other prokaryotic cells, somenucleic acids encoding proteins of interest can be modified to introducesolubility tags onto the protein of interest to ensure solubleexpression of the protein of interest. For example, addition of themaltose binding protein to a protein of interest has been shown toenhance soluble expression in E. coli [Sachdev, 1998; Kapust, 1999;Sachdev, 2000]. Either alternatively or in addition, chaperone proteins,such as DnaK, DnaJ, GroES and GroEL may be either co-expressed oroverexpressed with the proteins of interest, such as RuBisCO [Greene,2007], to promote correct folding and assembly [Martinez-Alonso, 2009;Martinez-Alonso, 2010].

For exogenous expression in Parococcus or other prokaryotic cells, somenucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acidscan encode targeting signals such as an N-terminal mitochondrial orother targeting signal, which can be removed before transformation intoprokaryotic host cells, if desired. For example, removal of amitochondrial leader sequence led to increased expression in E. coli[Hoffmeister, 2005]. For exogenous expression in yeast or othereukaryotic cells, genes can be expressed in the cytosol without theaddition of leader sequence, or can be targeted to mitochondrion orother organelles, or targeted for secretion, by the addition of asuitable targeting sequence such as a mitochondrial targeting orsecretion signal suitable for the host cells. Thus, it is understoodthat appropriate modifications to a nucleic acid sequence to remove orinclude a targeting sequence can be incorporated into an exogenousnucleic acid sequence to impart desirable properties.

Exemplary, optimized methods for introduction of exogenous nucleic acidsinto the methylotrophic bacteria Paracoccus versutus and Paracoccusdenitrificans via conjugative plasmid transfer are described in detailherein in Example 2.

Production of Central Metabolites as the Carbon-Based Products ofInterest

In certain embodiments, the engineered and/or evolved methylotroph ofthe present invention produces the central metabolites, including butnot limited to citrate, malate, succinate, fumarate, dihydroxyacetone,dihydroxyacetone phosphate, 3-hydroxypropionate, pyruvate, as thecarbon-based products of interest. The engineered and/or evolvedmethylotroph produces central metabolites as an intermediate or productof the carbon fixation or methylotrophic pathway or as a intermediate orproduct of host metabolism. In such cases, one or more transporters maybe expressed in the engineered and/or evolved methylotroph to export thecentral metabolite from the cell. For example, one or more members of afamily of enzymes known as C4-dicarboxylate carriers serve to exportsuccinate from cells into the media [Janausch, 2002; Kim, 2007]. Thesecentral metabolites can be converted to other products (FIG. 7).

In some embodiments, the engineered and/or evolved methylotroph mayinterconvert between different central metabolites to produce alternatecarbon-based products of interest. In one embodiment, the engineeredand/or evolved methylotroph produces aspartate by expressing one or moreaspartate aminotransferase (E.C. 2.6.1.1), such as Escherichia coliAspC, to convert oxaloacetate and L-glutamate to L-aspartate and2-oxoglutarate.

In another embodiment, the engineered and/or evolved methylotrophproduces dihydroxyacetone phosphate by expressing one or moredihydroxyacetone kinases (E.C. 2.7.1.29), such as C. freundii DhaK, toconvert dihydroxyacetone and ATP to dihydroxyacetone phosphate.

In another embodiment, the engineered and/or evolved methylotrophproduces serine as the carbon-based product of interest. The metabolicreactions necessary for serine biosynthesis include: phosphoglyceratedehydrogenase (E.C. 1.1.1.95), phosphoserine transaminase (E.C.2.6.1.52), phosphoserine phosphatase (E.C. 3.1.3.3). Phosphoglyceratedehydrogenase, such as E. coli SerA, converts 3-phospho-D-glycerate andNAD⁺ to 3-phosphonooxypyruvate and NADH. Phosphoserine transaminase,such as E. coli SerC, interconverts between3-phosphonooxypyruvate+L-glutamate andO-phospho-L-serine+2-oxoglutarate. Phosphoserine phosphatase, such as E.coli SerB, converts O-phospho-L-serine to L-serine.

In another embodiment, the engineered and/or evolved methylotrophproduces glutamate as the carbon-based product of interest. Themetabolic reactions necessary for glutamate biosynthesis includeglutamate dehydrogenase (E.C. 1.4.1.4; e.g., E. coli GdhA) whichconverts α-ketoglutarate, NH₃ and NADPH to glutamate. Glutamate cansubsequently be converted to various other carbon-based products ofinterest, e.g., according to the scheme presented in FIG. 8.

In another embodiment, the engineered and/or evolved methylotrophproduces itaconate as the carbon-based product of interest. Themetabolic reactions necessary for itaconate biosynthesis includeaconitate decarboxylase (E.C. 4.1.1.6; such as that from A. terreus)which converts cis-aconitate to itaconate and CO₂. Itaconate cansubsequently be converted to various other carbon-based products ofinterest, e.g., according to the scheme presented in FIG. 8.

Production of Sugars as the Carbon-Based Products of Interest

Industrial production of chemical products from biological organisms isoften accomplished using a sugar source, such as glucose or fructose, asthe feedstock. Hence, in certain embodiments, the engineered and/orevolved methylotroph of the present invention produces sugars includingglucose and fructose or sugar phosphates including triose phosphates(such as 3-phosphoglyceraldehyde and dihydroxyacetone-phosphate) as thecarbon-based products of interest. Sugars and sugar phosphates may alsobe interconverted. For example, glucose-6-phosphate isomerase (E.C.5.3.1.9; e.g., E. coli Pgi) may interconvert between D-fructose6-phosphate and D-glucose-6-phosphate. Phosphoglucomutase (E.C. 5.4.2.2;e.g., E. coli Pgm) converts D-α-glucose-6-P to D-α-glucose-1-P.Glucose-1-phosphatase (E.C. 3.1.3.10; e.g., E. coli Agp) convertsD-α-glucose-1-P to D-α-glucose. Aldose 1-epimerase (E.C. 5.1.3.3; e.g.,E. coli GalM) D-β-glucose to D-α-glucose. The sugars or sugar phosphatesmay optionally be exported from the engineered and/or evolvedmethylotroph into the culture medium.

Sugar phosphates may be converted to their corresponding sugars viadephosphorylation that occurs either intra- or extracellularly. Forexample, phosphatases such as a glucose-6-phosphatase (E.C. 3.1.3.9) orglucose−1-phosphatase (E.C. 3.1.3.10) can be introduced into theengineered and/or evolved methylotroph of the present invention.Exemplary phosphatases include Homo sapiens glucose-6-phosphatase G6PC(P35575), Escherichia coli glucose-1-phosphatase Agp (P19926), E.cloacae glucose-1-phosphatase AgpE (Q6EV19) and Escherichia coli acidphosphatase YihX (POA8Y3).

Sugar phosphates can be exported from the engineered and/or evolvedmethylotroph into the culture media via transporters. Transporters forsugar phosphates generally act as anti-porters with inorganic phosphate.An exemplary triose phosphate transporter includes A. thalianatriose-phosphate transporter APE2 (Genbank accession AT5G46110.4).Exemplary glucose-6-phosphate transporters include E. coli sugarphosphate transporter UhpT (NP_(—)418122.1), A. thalianaglucose-6-phosphate transporter GPT1 (AT5G54800.1), A. thalianaglucose-6-phosphate transporter GPT2, or homologs thereof.Dephosphorylation of glucose-6-phosphate can also be coupled to glucosetransport, such as Genbank accession numbers AAA16222, AAD19898, 043826.

Sugars can be diffusively effluxed from the engineered and/or evolvedmethylotroph into the culture media via permeases. Exemplary permeasesinclude H. sapiens glucose transporter GLUT-1, -3, or -7 (P11166,P11169, Q6PXP3), S. cerevisiae hexose transporter HXT-1, -4, or -6(P32465, P32467, P39003), Z. mobilis glucose uniporter Glf (P21906),Synechocystis sp. 1148 glucose/fructose:H⁺ symporter G1 cP (T.C.2.A.1.1.32; P15729) [Zhang, 1989], Streptomyces lividans major glucose(or 2-deoxyglucose) uptake transporter G1 cP (T.C. 2.A.1.1.35; Q7BEC4)[van Wezel, 2005], Plasmodium falciparum hexose (glucose and fructose)transporter PfHT1 (T.C. 2.A.1.1.24; 097467), or homologs thereof.Alternatively, to enable active efflux of sugars from the engineeredand/or evolved methylotroph, one or more active transporters may beintroduced to the cell. Exemplary transporters include mouse glucosetransporter GLUT 1 (AAB20846) or homologs thereof.

In some embodiments, to prevent buildup of other storage polymers fromsugars or sugar phosphates, the engineered and/or evolved methylotrophsof the present invention are attenuated in their ability to build otherstorage polymers such as glycogen, starch, sucrose, and cellulose usingone or more of the following enzymes: cellulose synthase (UDP forming)(E.C. 2.4.1.12), glycogen synthase e.g. glgA1, glgA2 (E.C. 2.4.1.21),sucrose phosphate synthase (E.C. 2.4.1.14), sucrose phosphorylase (E.C.3.1.3.24), alpha-1,4-glucan lyase (E.C. 4.2.2.13), glycogen synthase(E.C. 2.4.1.11), 1,4-alpha-glucan branching enzyme (E.C. 2.4.1.18).

The invention also provides engineered and/or evolved methylotrophs thatproduce other sugars such as sucrose, xylose, lactose, maltose, pentose,rhamnose, galactose and arabinose according to the same principles. Apathway for galactose biosynthesis is shown (FIG. 9). The metabolicreactions in the galactose biosynthetic pathway are catalyzed by thefollowing enzymes: alpha-D-glucose-6-phosphate ketol-isomerase (E.C.5.3.1.9; e.g., Arabidopsis thaliana PGI1), D-mannose-6-phosphateketol-isomerase (E.C. 5.3.1.8; e.g., Arabidopsis thaliana DINS),D-mannose 6-phosphate 1,6-phosphomutase (E.C. 5.4.2.8; e.g., Arabidopsisthaliana ATPMM), mannose-1-phosphate guanylyltransferase (E.C. 2.7.7.22;e.g., Arabidopsis thaliana CYT), GDP-mannose 3,5-epimerase (E.C.5.1.3.18; e.g., Arabidopsis thaliana GME), galactose-1-phosphateguanylyltransferase (E.C. 2.7.n.n; e.g., Arabidopsis thaliana VTC2),L-galactose 1-phosphate phosphatase (E.C. 3.1.3.n; e.g., Arabidopsisthaliana VTC4). In one embodiment, the invention provides an engineeredand/or evolved methylotroph comprising one or more exogenous proteinsfrom the galactose biosynthetic pathway.

The invention also provides engineered and/or evolved methylotrophs thatproduce sugar alcohols, such as sorbitol, as the carbon-based product ofinterest. In certain embodiments, the engineered and/or evolvedmethylotroph produces D-sorbitol from D-α-glucose and NADPH via theenzyme polyol dehydrogenase (E.C. 1.1.1.21; e.g., Saccharomycescerevisiae GRE3).

The invention also provides engineered and/or evolved methylotrophs thatproduce sugar derivatives, such as ascorbate, as the carbon-basedproduct of interest. In certain embodiments, the engineered and/orevolved methylotroph produces ascorbate from galactose via the enzymesL-galactose dehydrogenase (E.C. 1.1.1.122; e.g., Arabidopsis thalianaAt4G33670) and L-galactonolactone oxidase (E.C. 1.3.3.12; e.g.,Saccharomyes cerevisiae ATGLDH). Optionally, a catalase (E.C. 1.11.1.6;e.g., E. coli KatE) may be included to convert the waste producehydrogen peroxide to molecular oxygen.

The fermentation products according to the above aspect of the inventionare sugars, which are exported into the media as a result of C1metabolism during methylotrophy. The sugars can also be reabsorbed laterand fermented, directly separated, or utilized by a co-culturedorganism. This approach has several advantages. First, the total amountof sugars the cell can handle is not limited by maximum intracellularconcentrations because the end-product is exported to the media. Second,by removing the sugars from the cell, the equilibria of methylotrophicreactions are pushed towards creating more sugar. Third, duringmethylotrophy, there is no need to push carbon flow towards glycolysis.Fourth, the sugars are potentially less toxic than the fermentationproducts that would be directly produced.

Methylotrophy may be followed by flux of carbon compounds to thecreation and maintenance of biomass and to the storage of retrievablecarbon in the form of glycogen, cellulose and/or sucrose. Glycogen is apolymer of glucose composed of linear alpha 1,4-linkages and branchedalpha 1,6-linkages. The polymer is insoluble at degree of polymerization(DP) greater than about 60,000 and forms intracellular granules.Glycogen in synthesized in vivo via a pathway originating from glucose1-phosphate. Its hydrolysis can proceed through phosphorylation toglucose phosphates; via the internal cleavage of polymer tomaltodextrins; via the successive exo-cleavage to maltose; or via theconcerted hydrolysis of polymer and maltodextrins to maltose andglucose. Hence, an alternative biosynthetic route to glucose and/ormaltose is via the hydrolysis of glycogen which can optionally beexported from the cell as described above. There are a number ofpotential enzyme candidates for glycogen hydrolysis (Table 1).

In addition to the above, another mechanism is described to produceglucose biosynthetically. In certain embodiments, the present inventionprovides for cloned genes for glycogen hydrolyzing enzymes to hydrolyzeglycogen to glucose and/or maltose and transport maltose and glucosefrom the cell. Exemplary enzymes are set forth below in Table 1. Glucoseis transported from the engineered and/or evolved methylotroph by aglucose/hexose transporter. This alternative allows the cell toaccumulate glycogen naturally but adds enzyme activities to continuouslyreturn it to maltose or glucose units that can be collected as acarbon-based product.

TABLE 1 Enzymes for hydrolysis of glycogen E.C. Enzyme number Functionα-amylase 3.2.1.1 endohydrolysis of 1,4-α-D-glucosidic linkages inpolysaccharides β-amylase 3.2.1.2 hydrolysis of 1,4-α-D-glucosidiclinkages in polysaccharides so as to remove successive maltose unitsfrom the non-reducing ends of the chains γ-amylase 3.2.1.3 hydrolysis ofterminal 1,4-linked α-D-glucose residues successively from non-reducingends of the chains with release of β-D-glucose glucoamylase 3.2.1.3hydrolysis of terminal 1,4-linked α-D-glucose residues successively fromnon-reducing ends of the chains with release of β-D-glucose isoamylase3.2.1.68 hydrolysis of (1->6)-α-D-glucosidic branch linkages inglycogen, amylopectin and their beta-limit dextrins pullulanase 3.2.1.41hydrolysis of (1->6)-α-D-glucosidic linkages in pullulan [a linearpolymer of α-(1->6)-linked maltotriose units] and in amylopectin andglycogen, and the α- and β-limit dextrins of amylopectin and glycogenamylomaltase 2.4.1.25 transfers a segment of a 1,4-α-D-glucan to a newposition in an acceptor, which may be glucose or a 1,4-α-D-glucan (partof yeast debranching system) amylo-α-1,6- 3.2.1.33 debranching enzyme;hydrolysis of (1->6)-α-D-glucosidic branch linkages in glucosidaseglycogen phosphorylase limit dextrin phosphorylase 2.7.11.19 2 ATP +phosphorylase b = 2 ADP + phosphorylase a kinase phosphorylase 2.4.1.1(1,4-α-D-glucosyl)_(n) + phosphate = (1,4-α-D-glucosyl)_(n−1) +α-D-glucose-1-phosphate

Production of Fermentative Products as the Carbon-Based Products ofInterest

In certain embodiments, the engineered and/or evolved methylotroph ofthe present invention produces alcohols such as ethanol, propanol,isopropanol, butanol and fatty alcohols as the carbon-based products ofinterest.

In some embodiments, the engineered and/or evolved methylotroph of thepresent invention is engineered to produce ethanol via pyruvatefermentation. Pyruvate fermentation to ethanol is well know to those inthe art and there are several pathways including the pyruvatedecarboxylase pathway, the pyruvate synthase pathway and the pyruvateformate-lyase pathway (FIG. 10). The reactions in the pyruvatedecarboxylase pathway are catalyzed by the following enzymes: pyruvatedecarboxylase (E.C. 4.1.1.1) and alcohol dehydrogenase (E.C. 1.1.1.1 orE.C. 1.1.1.2). The reactions in the pyruvate synthase pathway arecatalyzed by the following enzymes: pyruvate synthase (E.C. 1.2.7.1),acetaldehyde dehydrogenase (E.C. 1.2.1.10 or E.C. 1.2.1.5), and alcoholdehydrogenase (E.C. 1.1.1.1 or E.C. 1.1.1.2). The reactions in thepyruvate formate-lyase pathway are catalyzed by the following enzymes:pyruvate formate-lyase (E.C. 2.3.1.54), acetaldehyde dehydrogenase (E.C.1.2.1.10 or E.C. 1.2.1.5), and alcohol dehydrogenase (E.C. 1.1.1.1 orE.C. 1.1.1.2).

In some embodiments, the engineered and/or evolved methylotroph of thepresent invention is engineered to produce lactate via pyruvatefermentation. Lactate dehydrogenase (E.C. 1.1.1.28) converts NADH andpyruvate to D-lactate. Exemplary enzymes include E. coli ldhA.

Currently, fermentative products such as ethanol, butanol, lactic acid,formate, acetate produced in biological organisms employ aNADH-dependent processes. However, depending on the metabolism of theengineered and/or evolved methylotroph, the cell may produce NADPH orreduced ferredoxin as the reducing cofactor. NADPH is used mostly forbiosynthetic operations in biological organisms, e.g., cell for growth,division, and for building up chemical stores, such as glycogen,sucrose, and other macromolecules. Using natural or engineered enzymesthat utilize NADPH or reduced ferredoxin as a source of reducing powerinstead of NADH would allow direct use of methylotrophic reducing powertowards formation of normally fermentative byproducts. Accordingly, thepresent invention provides methods for producing fermentative productssuch as ethanol by expressing NADP⁺-dependent or ferredoxin-dependentenzymes. NADP⁺-dependent enzymes include alcohol dehydrogenase [NADP⁺](E.C. 1.1.1.2) and acetaldehyde dehydrogenase [NAD(P)⁺] (E.C. 1.2.1.5).Exemplary NADP⁺-dependent alcohol dehydrogenases include Moorella sp.HUC22-1 AdhA (YP_(—)430754) [Inokuma, 2007], and homologs thereof.

In addition to providing exogenous genes or endogenous genes with novelregulation, the optimization of ethanol production in engineered and/orevolved methylotrophs sometimes requires the elimination or attenuationof certain host enzyme activities. These include, but are not limitedto, pyruvate oxidase (E.C. 1.2.2.2), D-lactate dehydrogenase (E.C.1.1.1.28), acetate kinase (E.C. 2.7.2.1), phosphate acetyltransferase(E.C. 2.3.1.8), citrate synthase (E.C. 2.3.3.1), phosphoenolpyruvatecarboxylase (E.C. 4.1.1.31). The extent to which these manipulations arenecessary is determined by the observed byproducts found in thebioreactor or shake-flask. For instance, observation of acetate wouldsuggest deletion of pyruvate oxidase, acetate kinase, and/orphosphotransacetylase enzyme activities. In another example, observationof D-lactate would suggest deletion of D-lactate dehydrogenase enzymeactivities, whereas observation of succinate, malate, fumarate,oxaloacetate, or citrate would suggest deletion of citrate synthaseand/or PEP carboxylase enzyme activities.

Production of Ethylene, Propylene, 1-Butene, 1,3-Butadiene, AcrylicAcid, Etc. as the Carbon-Based Products of Interest

In certain embodiments, the engineered and/or evolved methylotroph ofthe present invention produces ethylene, propylene, 1-butene,1,3-butadiene and acrylic acid as the carbon-based products of interest.Ethylene and/or propylene may be produced by either (1) the dehydrationof ethanol or propanol (E.C. 4.2.1.-), respectively or (2) thedecarboxylation of acrylate or crotonate (E.C. 4.1.1.-), respectively.While many dehydratases exist in nature, none has been shown to convertethanol to ethylene (or propanol to propylene, propionic acid to acrylicacid, etc.) by dehydration. Genes encoding enzymes in the 4.2.1.x or4.1.1.x group can be identified by searching databases such as GenBankusing the methods described above, expressed in any desired host (suchas Escherichia coli, for simplicity), and that host can be assayed forthe the appropriate enzymatic activity. A high-throughput screen isespecially useful for screening many genes and variants of genesgenerated by mutagenesis (i.e., error-prone PCR, synthetic libraries,chemical mutagenesis, etc.).

The ethanol dehydratase gene, after development to a suitable level ofactivity, can then be expressed in an ethanologenic organism to enablethat organism to produce ethylene. For instance, coexpress native orevolved ethanol dehydratase gene into an organism that already producesethanol, then test a culture by GC analysis of offgas for ethyleneproduction that is significantly higher than without the added gene orvia a high-throughput assay adapted from a colorimetric test [Larue,1973]. It may be desirable to eliminate ethanol-export proteins from theproduction organism to prevent ethanol from being secreted into themedium and preventing its conversion to ethylene.

Alternatively, acryloyl-CoA can be produced as described above, andacryloyl-CoA hydrolases (E.C. 3.1.2.-), such as the acuN gene fromHalomonas sp. HTNK1, can convert acryloyl-CoA into acrylate, which canbe thermally decarboxylated to yield ethylene.

Alternatively, genes encoding ethylene-forming enzyme activities (EfE,E.C. 1.14.17.4) from various sources are expressed. Exemplary enzymesinclude Pseudomonas syringae pv. Phaseolicola (BAA02477), P. syringaepv. Pisi (AAD16443), Ralstonia solanacearum (CAD18680). Optimizingproduction may require further metabolic engineering (improvingproduction of alpha-ketogluterate, recycling succinate as two examples).

In some embodiments, the engineered and/or evolved methylotroph of thepresent invention is engineered to produce ethylene from methionine. Thereactions in the ethylene biosynthesis pathway are catalyzed by thefollowing enzymes: methionine adenosyltransferase (E.C. 2.5.1.6),1-aminocyclopropane-1-carboxylate synthase (E.C. 4.4.1.14) and1-aminocyclopropane-1-carboxylate oxidase (E.C. 1.14.17.4).

In some embodiments, the engineered and/or evolved methylotroph of thepresent invention is engineered to produce propylene as the carbon-basedproduct of interest. In one embodiment, the engineered and/or evolvedmethylotroph is engineered to express one or more of the followingenzymes: propionyl-CoA synthase (E.C. 6.2.1.-, E.C. 4.2.1.- and E.C.1.3.1.-), propionyl-CoA transferase (E.C. 2.8.3.1), aldehydedehydrogenase (E.C. 1.2.1.3 or E.C. 1.2.1.4), alcohol dehydrogenase(E.C. 1.1.1.1 or E.C. 1.1.1.2), and alcohol dehydratase (E.C. 4.2.1.-).Propionyl-CoA synthase is a multi-functional enzyme that converts3-hydroxypropionate, ATP and NADPH to propionyl-CoA. Exemplarypropionyl-CoA synthases include AAL47820, and homologs thereof. Thepresent invention provides nucleic acids each comprising or consistingof a sequence which is a codon optimized version of the wild-typepropionyl-CoA synthase gene. In another embodiment, the inventionprovides a nucleic acid encoding a polypeptide having the amino acidsequence of SEQ ID NO:5, or a sequence having 70%, %, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81-85%, 90-95%, 96-98%, 99%, 99.9% or evenhigher identity thereto. Propionyl-CoA transferase convertspropionyl-CoA and acetate to acetyl-CoA and propionate. Exemplaryenzymes include Ralstonia eutropha pct and homologs thereof. Aldehydedehydrogenase converts propionate and NADPH to propanal. Alcoholdehydrogenase converts propanal and NADPH to 1-propanol. Alcoholdehydratase converts 1-propanol to propylene.

In another embodiment, E. coli thiolase atoB (E.C. 2.3.1.9) converts 2acetyl-CoA into acetoacetyl-CoA, and C. acetobutylicum hbd (E.C.1.1.1.157) converts acetoacetyl-CoA and NADH into 3-hydroxybutyryl-CoA.E. coli tesB (EC 3.1.2.20) or C. acetobutylicum ptb and buk (E.C.2.3.1.19 and 2.7.2.7 respectively) convert 3-hydroxybutyryl-CoA into3-hydroxybutyrate, which can be simultaneously decarboxylated anddehydrated to yield propylene. Optionally, the 3-hydroxybutyryl-CoA ispolymerized to form poly(3-hydroxybutyrate), a solid compound which canbe extracted from the fermentation medium and simultaneouslydepolymerizied, hydrolyzed, dehydrated, and decarboxyated to yieldpropylene (U.S. patent application Ser. No. 12/527,714, 2008).

Production of Fatty Acids, their Intermediates and Derivatives as theCarbon-Based Products of Interest

In certain embodiments, the engineered and/or evolved methylotroph ofthe present invention produces fatty acids, their intermediates andtheir derivatives as the carbon-based products of interest. Theengineered and/or evolved methylotrophs of the present invention can bemodified to increase the production of acyl-ACP or acyl-CoA, to reducethe catabolism of fatty acid derivatives and intermediates, or to reducefeedback inhibition at specific points in the biosynthetic pathway usedfor fatty acid products. In addition to modifying the genes describedherein, additional cellular resources can be diverted to over-producefatty acids. For example the lactate, succinate and/or acetate pathwayscan be attenuated and the fatty acid biosynthetic pathway precursorsacetyl-CoA and/or malonyl-CoA can be overproduced.

In one embodiment, the engineered and/or evolved methylotrophs of thepresent invention can be engineered to express certain fatty acidsynthase activities (FAS), which is a group of peptides that catalyzethe initiation and elongation of acyl chains [Marrakchi, 2002a]. Theacyl carrier protein (ACP) and the enzymes in the FAS pathway controlthe length, degree of saturation and branching of the fatty acidsproduced, which can be attenuated or over-expressed. Such enzymesinclude accABCD, FabD, FabH, FabG, FabA, FabZ, Fabl, FabK, FabL, FabM,FabB, FabF, and homologs thereof.

In another embodiment, the engineered and/or evolved methylotrophs ofthe present invention form fatty acid byproducts through ACP-independentpathways, for example, the pathway described recently by [Dellomonaco,2011] involving reversal of beta oxidation. Enzymes involved in thesepathways include such genes as atoB, fadA, fadB, fadD, fadE, fad I,fadK, fadJ, paaZ, ydiO, yfcY, yfcZ, ydiD, and homologs thereof.

In one aspect, the fatty acid biosynthetic pathway precursors acetyl-CoAand malonyl-CoA can be overproduced in the engineered and/or evolvedmethylotroph of the present invention. Several different modificationscan be made, either in combination or individually, to the host cell toobtain increased acetyl CoA/malonyl CoA/fatty acid and fatty acidderivative production. To modify acetyl-CoA and/or malonyl-CoAproduction, the expression of acetyl-CoA carboxylase (E.C. 6.4.1.2) canbe modulated. Exemplary genes include accABCD (AAC73296) or homologsthereof. To increase acetyl CoA production, the expression of severalgenes may be altered including pdh, panK, aceEF, (encoding the Elpdehydrogenase component and the E2p dihydrolipoamide acyltransferasecomponent of the pyruvate and 2-oxoglutarate dehydrogenase complexes),fabH/fabD/fabG/acpP/fabF, and in some examples additional nucleic acidencoding fatty-acyl-CoA reductases and aldehyde decarbonylases.Exemplary enzymes include pdh (BAB34380, AAC73227, AAC73226), panK (alsoknown as coaA, AAC76952), aceEF (AAC73227, AAC73226), fabH (AAC74175),fabD (AAC74176), fabG (AAC74177), acpP (AAC74178), fabF (AAC74179).

Genes to be knocked-out or attenuated include fadE, gpsA, ldhA, pflb,adhE, pta, poxB, ackA, and/or ackB. Exemplary enzymes include fadE(AAC73325), gspA (AAC76632), ldhA (AAC74462), pflb (AAC73989), adhE(AAC74323), pta (AAC75357), poxB (AAC73958), ackA (AAC75356), ackB(BAB81430), and homologs thereof.

Additional potential modifications include the following. To achievefatty acid overproduction, lipase (E.C. 3.1.1.3) which producetriacylglyerides from fatty acids and glycerol and in some cases servesas a suppressor of fabA can be included in the engineered and/or evolvedmethylotroph of the present invention. Exemplary enzymes includeSaccharomyces cerevisiae LipA (CAA89087), Saccharomyces cerevisiae TGL2CAA98876, and homologs thereof. To remove limitations on the pool ofacyl-CoA, the D311E mutation in plsB (AAC77011) can be introduced.

To engineer an engineered and/or evolved methylotroph for the productionof a population of fatty acid derivatives with homogeneous chain length,one or more endogenous genes can be attenuated or functionally deletedand one or more thioesterases can be expressed. Thioesterases (E.C.3.1.2.14) generate acyl-ACP from fatty acid and ACP. For example, C10fatty acids can be produced by attenuating endogenous C18 thioesterases(for example, E. coli tesA AAC73596 and POADA1, and homologs thereof),which uses C18:1-ACP, and expressing a C10 thioesterase, which usesC10-ACP, thus, resulting in a relatively homogeneous population of fattyacids that have a carbon chain length of 10. In another example, C14fatty acid derivatives can be produced by attenuating endogenousthioesterases that produce non-C14 fatty acids and expressing the C14thioesterase, which uses C14-ACP. In yet another example, C12 fatty acidderivatives can be produced by expressing thioesterases that use C12-ACPand attenuating thioesterases that produce non-C12 fatty acids.Exemplary C8:0 to C10:0 thioesterases include Cuphea hookeriana fatB2(AAC49269) and homologs thereof. Exemplary C12:0 thioesterases includeUmbellularia california fatB (Q41635) and homologs thereof. ExemplaryC14:0 thioesterases include Cinnamonum camphorum fatB (Q39473).Exemplary C14:0 to C16:0 thioesterases include Cuphea hookeriana fatB3(AAC49269). Exemplary C16:0 thioesterases include Arabidopsis thalianafatB (CAA85388), Cuphea hookeriana fatB1 (Q39513) and homologs thereof.Exemplary C18:1 thioesterases include Arabidopsis thaliana fatA(NP_(—)189147, NP_(—)193041), Arabidopsis thaliana fatB (CAA85388),Bradyrhizobium japonicum fatA (CAC39106), Cuphea hookeriana fatA(AAC72883), Escherichia coli tesA (NP_(—)415027) and homologs thereof.Acetyl CoA, malonyl CoA, and fatty acid overproduction can be verifiedusing methods known in the art, for example by using radioactiveprecursors, HPLC, and GC-MS subsequent to cell lysis.

In yet another aspect, fatty acids of various lengths can be produced inthe engineered and/or evolved methylotroph by expressing oroverexpressing acyl-CoA synthase peptides (E.C. 2.3.1.86), whichcatalyzes the conversion of fatty acids to acyl-CoA. Some acyl-CoAsynthase peptides, which are non-specific, accept other substrates inaddition to fatty acids.

In yet another aspect, branched chain fatty acids, their intermediatesand their derivatives can be produced in the engineered and/or evolvedmethylotroph as the carbon-based products of interest. By controllingthe expression of endogenous and heterologous enzymes associated withbranched chain fatty acid biosynthesis, the production of branched chainfatty acid intermediates including branched chain fatty acids can beenhanced. Branched chain fatty acid production can be achieved throughthe expression of one or more of the following enzymes [Kaneda, 1991]:branched chain amino acid aminotransferase to produce α-ketoacids frombranched chain amino acids such as isoleucine, leucine and valine (E.C.2.6.1.42), branched chain α-ketoacid dehydrogenase complexes whichcatalyzes the oxidative decarboxylation of α-ketoacids to branched chainacyl-CoA (bkd, E.C. 1.2.4.4) [Denoya, 1995], dihydrolipoyl dehydrogenase(E.C. 1.8.1.4), beta-ketoacyl-ACP synthase with branched chain acyl CoAspecificity (E.C. 2.3.1.41) [Li, 2005], crotonyl-CoA reductase (E.C.1.3.1.8, 1.3.1.85 or 1.3.1.86) [Han, 1997], and isobutyryl-CoA mutase(large subunit E.C. 5.4.99.2 and small subunit E.C. 5.4.99.13).Exemplary branched chain amino acid aminotransferases include E. coliilvE (YP_(—)026247), Lactococcus lactis ilvE (AAF34406), Pseudomonasputida ilvE (NP_(—)745648), Streptomyces coelicolor ilvE (NP_(—)629657),and homologs thereof. Branched chain α-ketoacid dehydrogenase complexesconsist of E1α/β (decarboxylase), E2 (dihydrolipoyl transacylase) and E3(dihydrolipoyl dehydrogenase) subunits. The industrial host E. coli hasonly the E3 component as a part of its pyruvate dehydrogenase complex(lpd, E.C. 1.8.1.4, NP_(—)414658) and so it requires the E1α/β and E2bkd proteins. Exemplary α-ketoacid dehydrogenase complexes includeStreptomyces coelicolor bkdA1 (NP_(—)628006) E1α (decarboxylasecomponent), S. coelicolor bkdB2 (NP_(—)628005) E1β (decarboxylasecomponent), S. coelicolor bkdA3 (NP_(—)638004) E2 (dihydrolipoyltransacylase); or S. coelicolor bkdA2 (NP_(—)733618) E1α (decarboxylasecomponent), S. coelicolor bkdB2 (NP_(—)628019) E1β (decarboxylasecomponent), S. coelicolor bkdC2 (NP_(—)628018) E2 (dihydrolipoyltransacylase); or S. avermitilis bkdA (BAC72074) E1α (decarboxylasecomponent), S. avermitilis bkdB (BAC72075) E1β (decarboxylasecomponent), S. avermitilis bkdC (BAC72076) E2 (dihydrolipoyltransacylase); S. avermitilis bkdF (E.C.1.2.4.4, BAC72088) E1α(decarboxylase component), S. avermitilis bkdG (BAC72089) E1β(decarboxylase component), S. avermitilis bkdH (BAC72090) E2(dihydrolipoyl transacylase); B. subtilis bkdAA (NP_(—)390288) E1α(decarboxylase component), B. subtilis bkdAB (NP_(—)390288) E1β(decarboxylase component), B. subtilis bkdB (NP_(—)390288) E2(dihydrolipoyl transacylase); or P. putida bkdA1 (AAA65614) Ela(decarboxylase component), P. putida bkdA2 (AAA65615) E1β (decarboxylasecomponent), P. putida bkdC (AAA65617) E2 (dihydrolipoyl transacylase);and homologs thereof. An exemplary dihydrolipoyl dehydrogenase is E.coli lpd (NP_(—)414658) E3 and homologs thereof. Exemplarybeta-ketoacyl-ACP synthases with branched chain acyl CoA specificityinclude Streptomyces coelicolor fabH1 (NP_(—)626634), ACP (NP_(—)626635)and fabF (NP_(—)626636); Streptomyces avermitilis fabH3 (NP_(—)823466),fabC3 (NP_(—)823467), fabF (NP_(—)823468); Bacillus subtilis fabH_A(NP_(—)389015), fabH_B (NP_(—)388898), ACP (NP_(—)389474), fabF(NP_(—)389016); Stenotrophomonas maltophilia SmalDRAFT_(—)0818(ZP_(—)01643059), SmalDRAFT_(—)0821 (ZP_(—)01643063), SmalDRAFT_(—)0822(ZP_(—)01643064); Legionella pneumophila fabH (YP_(—)123672), ACP(YP_(—)123675), fabF (YP_(—)123676); and homologs thereof. Exemplarycrotonyl-CoA reductases include Streptomyces coelicolor ccr(NP_(—)630556), Streptomyces cinnamonensis ccr (AAD53915), and homologsthereof. Exemplary isobutyryl-CoA mutases include Streptomycescoelicolor icmA & icmB (NP_(—)629554 and NP_(—)630904), Streptomycescinnamonensis icmA and icmB (AAC08713 and AJ246005), and homologsthereof. Additionally or alternatively, endogenous genes that normallylead to straight chain fatty acids, their intermediates, and derivativesmay be attenuated or deleted to eliminate competing pathways. Enzymesthat interfere with production of branched chain fatty acids includeβ-ketoacyl-ACP synthase II (E.C. 2.3.1.41) and β-ketoacyl-ACP synthaseIII (E.C. 2.3.1.41) with straight chain acyl CoA specificity. Exemplaryenzymes for deletion include E. coli fabF (NP_(—)415613) and fabH(NP_(—)415609).

In yet another aspect, fatty acids, their intermediates and theirderivatives with varying degrees of saturation can be produced in theengineered and/or evolved methylotroph as the carbon-based products ofinterest. In one aspect, hosts are engineered to produce unsaturatedfatty acids by over-expressing β-ketoacyl-ACP synthase I (E.C.2.3.1.41), or by growing the host at low temperatures (for example lessthan 37° C.). FabB has preference to cis-δ³decenoyl-ACP and results inunsaturated fatty acid production in E. coli. Over-expression of FabBresults in the production of a significant percentage of unsaturatedfatty acids [de Mendoza, 1983]. These unsaturated fatty acids can thenbe used as intermediates in hosts that are engineered to produce fattyacids derivatives, such as fatty alcohols, esters, waxes, olefins,alkanes, and the like. Alternatively, the repressor of fatty acidbiosynthesis, E. coli FabR (NP_(—)418398), can be deleted, which canalso result in increased unsaturated fatty acid production in E. coli[Zhang, 2002]. Further increase in unsaturated fatty acids is achievedby over-expression of heterologous trans-2, cis-3-decenoyl-ACP isomeraseand controlled expression of trans-2-enoyl-ACP reductase II [Marrakchi,2002b], while deleting E. coli FabI (trans-2-enoyl-ACP reductase, E.C.1.3.1.9, NP_(—)415804) or homologs thereof in the host organism.Exemplary β-ketoacyl-ACP synthase I include Escherichia coli fabB(BAA16180) and homologs thereof. Exemplary trans-2, cis-3-decenoyl-ACPisomerase include Streptococcus mutans UA159 FabM (DAA05501) andhomologs thereof. Exemplary trans-2-enoyl-ACP reductase II includeStreptococcus pneumoniae R6 FabK (NP_(—)357969) and homologs thereof. Toincrease production of monounsaturated fatty acids, the sfa gene,suppressor of FabA, can be over-expressed [Rock, 1996]. Exemplaryproteins include AAN79592 and homologs thereof. One of ordinary skill inthe art would appreciate that by attenuating fabA, or over-expressingfabB and expressing specific thioesterases (described above),unsaturated fatty acids, their derivatives, and products having adesired carbon chain length can be produced.

In some examples the fatty acid or intermediate is produced in thecytoplasm of the cell. The cytoplasmic concentration can be increased ina number of ways, including, but not limited to, binding of the fattyacid to coenzyme A to form an acyl-CoA thioester. Additionally, theconcentration of acyl-CoAs can be increased by increasing thebiosynthesis of CoA in the cell, such as by over-expressing genesassociated with pantothenate biosynthesis (panD) or knocking out thegenes associated with glutathione biosynthesis (glutathione synthase).

Production of Fatty Alcohols as the Carbon-Based Products of Interest

In yet further aspects, hosts cells are engineered to convert acyl-CoAto fatty alcohols by expressing or overexpressing a fatty alcoholforming acyl-CoA reductase (FAR, E.C. 1.1.1.*), or an acyl-CoAreductases (E.C. 1.2.1.50) and alcohol dehydrogenase (E.C. 1.1.1.1) or acombination of the foregoing to produce fatty alcohols from acyl-CoA.Hereinafter fatty alcohol forming acyl-CoA reductase (FAR, E.C.1.1.1.*), acyl-CoA reductases (E.C. 1.2.1.50) and alcohol dehydrogenase(E.C. 1.1.1.1) are collectively referred to as fatty alcohol formingpeptides. Some fatty alcohol forming peptides are non-specific andcatalyze other reactions as well: for example, some acyl-CoA reductasepeptides accept other substrates in addition to fatty acids. Exemplaryfatty alcohol forming acyl-CoA reductases include Acinetobacter baylyiADP1 acrl (AAC45217), Simmondsia chinensis jjfar (AAD38039), Musmusculus mfar1 (AAH07178), Mus musculus mfar2 (AAH55759), Acinetobactersp. M1 acrM1, Homo sapiens hfar (AAT42129), and homologs thereof. Fattyalcohols can be used as surfactants.

Many fatty alcohols are derived from the products of fatty acidbiosynthesis. Hence, the production of fatty alcohols can be controlledby engineering fatty acid biosynthesis in the engineered and/or evolvedmethylotroph. The chain length, branching and degree of saturation offatty acids and their intermediates can be altered using the methodsdescribed herein, thereby affecting the nature of the resulting fattyalcohols.

As mentioned above, through the combination of expressing genes thatsupport brFA synthesis and alcohol synthesis, branched chain alcoholscan be produced. For example, when an alcohol reductase such as Acrlfrom Acinetobacter baylyi ADP1 is coexpressed with a bkd operon, E. colican synthesize isopentanol, isobutanol or 2-methyl butanol. Similarly,when Acrl is coexpressed with ccr/icm genes, E. coli can synthesizeisobutanol.

Production of Fatty Esters as the Carbon-Based Products of Interest

In another aspect, engineered and/or evolved methylotrophs producevarious lengths of fatty esters (biodiesel and waxes) as thecarbon-based products of interest. Fatty esters can be produced fromacyl-CoAs and alcohols. The alcohols can be provided in the fermentationmedia, produced by the engineered and/or evolved methylotroph itself orproduced by a co-cultured organism.

In some embodiments, one or more alcohol O-acetyltransferases isexpressed in the engineered and/or evolved methylotroph to produce fattyesters as the carbon-based product of interest. AlcoholO-acetyltransferase (E.C. 2.3.1.84) catalyzes the reaction of acetyl-CoAand an alcohol to produce CoA and an acetic ester. In some embodiments,the alcohol O-acetyltransferase peptides are co-expressed with selectedthioesterase peptides, FAS peptides and fatty alcohol forming peptidesto allow the carbon chain length, saturation and degree of branching tobe controlled. In other embodiments, the bkd operon can be co-expressedto enable branched fatty acid precursors to be produced.

Alcohol O-acetyltransferase peptides catalyze other reactions such thatthe peptides accept other substrates in addition to fatty alcohols oracetyl-CoA thioester. Other substrates include other alcohols and otheracyl-CoA thioesters. Modification of such enzymes and the development ofassays for characterizing the activity of a particular alcoholO-acetyltransferase peptides are within the scope of a skilled artisan.Engineered O-acetyltransferases and O-acyltransferases can be createdthat have new activities and specificities for the donor acyl group oracceptor alcohol moiety.

Alcohol acetyl transferases (AATs, E.C. 2.3.1.84), which are responsiblefor acyl acetate production in various plants, can be used to producemedium chain length waxes, such as octyl octanoate, decyl octanoate,decyl decanoate, and the like. Fatty esters, synthesized from mediumchain alcohol (such as C6, C8) and medium chain acyl-CoA (or fattyacids, such as C6 or C8) have a relative low melting point. For example,hexyl hexanoate has a melting point of −55° C. and octyl octanoate has amelting point of −18 to −17° C. The low melting points of thesecompounds make them good candidates for use as biofuels. Exemplaryalcohol acetyltransferases include Fragaria x ananassa SAAT (AAG13130)[Aharoni, 2000], Saccharomyces cerevisiae Atfpl (NP_(—)015022), andhomologs thereof.

In some embodiments, one or more wax synthases (E.C. 2.3.1.75) isexpressed in the engineered and/or evolved methylotroph to produce fattyesters including waxes from acyl-CoA and alcohols as the carbon-basedproduct of interest. Wax synthase peptides are capable of catalyzing theconversion of an acyl-thioester to fatty esters. Some wax synthasepeptides can catalyze other reactions, such as converting short chainacyl-CoAs and short chain alcohols to produce fatty esters. Methods toidentify wax synthase activity are provided in U.S. Pat. No. 7,118,896,which is herein incorporated by reference. Medium-chain waxes that havelow melting points, such as octyl octanoate and octyl decanoate, aregood candidates for biofuel to replace triglyceride-based biodiesel.Exemplary wax synthases include Acinetobacter baylyi ADP1 wsadp1,Acinetobacter baylyi ADP1 wax-dgaT (AA017391) [Kalscheuer, 2003],Saccharomyces cerevisiae Eeb1 (NP_(—)015230), Saccharomyces cerevisiaeYMR210w (NP_(—)013937), Simmondsia chinensis acyltransferase (AAD38041),Mus musculus Dgat214 (Q6E1M8), and homologs thereof.

In other aspects, the engineered and/or evolved methylotrophs aremodified to produce a fatty ester-based biofuel by expressing nucleicacids encoding one or more wax ester synthases in order to confer theability to synthesize a saturated, unsaturated, or branched fatty ester.In some embodiments, the wax ester synthesis proteins include, but arenot limited to: fatty acid elongases, acyl-CoA reductases,acyltransferases or wax synthases, fatty acyl transferases,diacylglycerol acyltransferases, acyl-coA wax alcohol acyltransferases,bifunctional wax ester synthase/acyl-CoA: diacylglycerol acyltransferaseselected from a multienzyme complex from Simmondsia chinensis,Acinetobacter sp. strain ADP1 (formerly Acinetobacter calcoaceticusADP1), Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsisthaliana, or Alkaligenes eutrophus. In one embodiment, the fatty acidelongases, acyl-CoA reductases or wax synthases are from a multienzymecomplex from Alkaligenes eutrophus and other organisms known in theliterature to produce wax and fatty acid esters.

Many fatty esters are derived from the intermediates and products offatty acid biosynthesis. Hence, the production of fatty esters can becontrolled by engineering fatty acid biosynthesis in the engineeredand/or evolved methylotroph. The chain length, branching and degree ofsaturation of fatty acids and their intermediates can be altered usingthe methods described herein, thereby affecting the nature of theresulting fatty esters.

Additionally, to increase the percentage of unsaturated fatty acidesters, the engineered and/or evolved methylotroph can also overexpressSfa which encodes a suppressor of fabA (AAN79592, AAC44390),β-ketoacyl-ACP synthase I (E.C. 2.3.1.41, BAA16180), and secG nullmutant suppressors (cold shock proteins) gnsA and gnsB (ABD18647 andAAC74076). In some examples, the endogenous fabF gene can be attenuated,thus, increasing the percentage of palmitoleate (C 16:1) produced.

Optionally a wax ester exporter such as a member of the FATP family isused to facilitate the release of waxes or esters into the extracellularenvironment from the engineered and/or evolved methylotroph. Anexemplary wax ester exporter that can be used is fatty acid (long chain)transport protein CG7400-PA, isoform A from D. melanogaster(NP_(—)524723), or homologs thereof.

The centane number (CN), viscosity, melting point, and heat ofcombustion for various fatty acid esters have been characterized in forexample, [Knothe, 2005]. Using the teachings provided herein theengineered and/or evolved methylotroph can be engineered to produce anyone of the fatty acid esters described in [Knothe, 2005].

Production of Alkanes as the Carbon-Based Products of Interest

In another aspect, engineered and/or evolved methylotrophs producealkanes of various chain lengths (hydrocarbons) as the carbon-basedproducts of interest. Many alkanes are derived from the products offatty acid biosynthesis. Hence, the production of alkanes can becontrolled by engineering fatty acid biosynthesis in the engineeredand/or evolved methylotroph. The chain length, branching and degree ofsaturation of fatty acids and their intermediates can be altered usingthe methods described herein. The chain length, branching and degree ofsaturation of alkanes can be controlled through their fatty acidbiosynthesis precursors.

In certain aspects, fatty aldehydes can be converted to alkanes and COin the engineered and/or evolved methylotroph via the expression ofdecarbonylases [Cheesbrough, 1984; Dennis, 1991]. Exemplary enzymesinclude Arabidopsis thaliana cerl (NP_(—)171723), Oryza sativacer1 CER1(AAD29719) and homologs thereof.

In another aspect, fatty alcohols can be converted to alkanes in theengineered and/or evolved methylotroph via the expression of terminalalcohol oxidoreductases as in Vibrio furnissii M1 [Park, 2005].

Production of Olefins as the Carbon-Based Products of Interest

In another aspect, engineered and/or evolved methylotrophs produceolefins (hydrocarbons) as the carbon-based products of interest. Olefinsare derived from the intermediates and products of fatty acidbiosynthesis. Hence, the production of olefins can be controlled byengineering fatty acid biosynthesis in the engineered and/or evolvedmethylotroph. Introduction of genes affecting the production ofunsaturated fatty acids, as described above, can result in theproduction of olefins. Similarly, the chain length of olefins can becontrolled by expressing, overexpressing or attenuating the expressionof endogenous and heterologous thioesterases which control the chainlength of the fatty acids that are precursors to olefin biosynthesis.Also, by controlling the expression of endogenous and heterologousenzymes associated with branched chain fatty acid biosynthesis, theproduction of branched chain olefins can be enhanced. Methods forcontrolling the chain length and branching of fatty acid biosynthesisintermediates and products are described above. Olefins can be obtainedby downstreaming processing of 3-hydroxy alkanoates as taught by Fischeret al. [Ind Eng Chem Res, 2011, 50(8):4420-4424, DOI:10.1021/ie1023386]. Accordingly, the fermentation product formethylotrophic production of olefins need not be an olefin itself.

Production of ω-Cyclic Fatty Acids and their Derivatives as theCarbon-Based Products of Interest

In another aspect, the engineered and/or evolved methylotroph of thepresent invention produces co-cyclic fatty acids (cyFAs) as thecarbon-based product of interest. To synthesize co-cyclic fatty acids(cyFAs), several genes need to be introduced and expressed that providethe cyclic precursor cyclohexylcarbonyl-CoA [Cropp, 2000]. The genes(fabH, ACP and fabF) can then be expressed to allow initiation andelongation of co-cyclic fatty acids. Alternatively, the homologous genescan be isolated from microorganisms that make cyFAs and expressed in E.coli. Relevant genes include bkdC, lpd, fabH, ACP, fabF, fabH1, ACP,fabF, fabH3, fabC3, fabF, fabH_A, fabH_B, ACP.

Expression of the following genes are sufficient to providecyclohexylcarbonyl-CoA in E. coli: ansJ, ansK, ansL, chcA(1-cyclohexenylcarbonyl CoA reductase) and ansM from the ansatrieningene cluster of Streptomyces collinus [Chen, 1999] or plmJK(5-enolpyruvylshikimate-3-phosphate synthase), plmL (acyl-CoAdehydrogenase), chcA (enoyl-(ACP) reductase) and plmM (2,4-dienoyl-CoAreductase) from the phoslactomycin B gene cluster of Streptomyces sp.HK803 [Palaniappan, 2003] together with the acyl-CoA isomerase (chcBgene) [Patton, 2000] from S. collinus, S. avermitilis or S. coelicolor.Exemplary ansatrienin gene cluster enzymes include AAC44655, AAF73478and homologs thereof. Exemplary phoslactomycin B gene cluster enzymesinclude AAQ84158, AAQ84159, AAQ84160, AAQ84161 and homologs thereof.Exemplary chcB enzymes include NP_(—)629292, AAF73478 and homologsthereof.

The genes (fabH, ACP and fabF) are sufficient to allow initiation andelongation of co-cyclic fatty acids, because they can have broadsubstrate specificity. In the event that coexpression of any of thesegenes with the ansJKLM/chcAB or pmlJKLM/chcAB genes does not yieldcyFAs, fabH, ACP and/or fabF homologs from microorganisms that makecyFAs can be isolated (e.g., by using degenerate PCR primers orheterologous DNA probes) and coexpressed.

Production of Halogenated Derivatives of Fatty Acids

Genes are known that can produce fluoroacetyl-CoA from fluoride ion. Inone embodiment, the present invention allows for production offluorinated fatty acids by combining expression offluoroacetate-involved genes (e.g., fluorinase, nucleotidephosphorylase, fluorometabolite-specific aldolases, fluoroacetaldehydedehydrogenase, and fluoroacetyl-CoA synthase).

Transport/Efflux/Release of Fatty Acids and their Derivatives

Also disclosed herein is a system for continuously producing andexporting hydrocarbons out of recombinant host microorganisms via atransport protein. Many transport and efflux proteins serve to excrete alarge variety of compounds and can be evolved to be selective for aparticular type of fatty acid. Thus, in some embodiments an ABCtransporter can be functionally expressed by the engineered and/orevolved methylotroph, so that the organism exports the fatty acid intothe culture medium. In one example, the ABC transporter is an ABCtransporter from Caenorhabditis elegans, Arabidopsis thalania,Alkaligenes eutrophus or Rhodococcus erythropolis or homologs thereof.Exemplary transporters include AAU44368, NP_(—)188746, NP_(—)175557,AAN73268 or homologs thereof.

The transport protein, for example, can also be an efflux proteinselected from: AcrAB (NP_(—)414996.1, NP_(—)414995.1), ToIC(NP_(—)417507.2) and AcrEF (NP_(—)417731.1, NP_(—)417732.1) from E.coli, or t111618 (NP_(—)682408), t111619 (NP_(—)682409), t110139(NP_(—)680930), H11619 and U10139 from Thermosynechococcus elongatusBP-I or homologs thereof.

In addition, the transport protein can be, for example, a fatty acidtransport protein (FATP) selected from Drosophila melanogaster,Caenorhabditis elegans, Mycobacterium tuberculosis or Saccharomycescerevisiae, Acinetobacter sp. H01-N, any one of the mammalian FATPs orhomologs thereof. The FATPs can additionally be resynthesized with themembranous regions reversed in order to invert the direction ofsubstrate flow. Specifically, the sequences of amino acids composing thehydrophilic domains (or membrane domains) of the protein can be invertedwhile maintaining the same codons for each particular amino acid. Theidentification of these regions is well known in the art.

Production of Isoprenoids as the Carbon-Based Products of Interest

In one aspect, the engineered and/or evolved methylotroph of the presentinvention produces isoprenoids or their precursors isopentenylpyrophosphate (IPP) and its isomer, dimethylallyl pyrophosphate (DMAPP)as the carbon-based products of interest. There are two knownbiosynthetic pathways that synthesize IPP and DMAPP. Prokaryotes, withsome exceptions, use the mevalonate-independent or deoxyxylulose5-phosphate (DXP) pathway to produce IPP and DMAPP separately through abranch point (FIG. 11). Eukaryotes other than plants use themevalonate-dependent (MEV) isoprenoid pathway exclusively to convertacetyl-coenzyme A (acetyl-CoA) to IPP, which is subsequently isomerizedto DMAPP (FIG. 12). In general, plants use both the MEV and DXP pathwaysfor IPP synthesis.

The reactions in the DXP pathway are catalyzed by the following enzymes:1-deoxy-D-xylulose-5-phosphate synthase (E.C. 2.2.1.7),1-deoxy-D-xylulose-5-phosphate reductoisomerase (E.C. 1.1.1.267),4-diphosphocytidyl-2C-methyl-D-erythritol synthase (E.C. 2.7.7.60),4-diphosphocytidyl-2C-methyl-D-erythritol kinase (E.C. 2.7.1.148),2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (E.C. 4.6.1.12),(E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (E.C. 1.17.7.1),isopentyl/dimethylallyl diphosphate synthase or4-hydroxy-3-methylbut-2-enyl diphosphate reductase (E.C. 1.17.1.2). Inone embodiment, the engineered and/or evolved methylotroph of thepresent invention expresses one or more enzymes from the DXP pathway.For example, one or more exogenous proteins can be selected from1-deoxy-D-xylulose-5-phosphate reductoisomerase,4-diphosphocytidyl-2C-methyl-D-erythritol synthase,4-diphosphocytidyl-2C-methyl-D-erythritol kinase, 2C-methyl-D-erythritol2,4-cyclodiphosphate synthase, (E)-4-hydroxy-3-methylbut-2-enyldiphosphate synthase, and 4-hydroxy-3-methylbut-2-enyl diphosphatereductase. The host organism can also express two or more, three ormore, four or more, and the like, including up to all the protein andenzymes that confer the DXP pathway. Exemplary1-deoxy-D-xylulose-5-phosphate synthases include E. coli Dxs (AAC46162);P. putida KT2440 Dxs (AAN66154); Salmonella enterica Paratyphi, see ATCC9150 Dxs (AAV78186); Rhodobacter sphaeroides 2.4.1 Dxs (YP_(—)353327);Rhodopseudomonas palustris CGA009 Dxs (NP_(—)946305); Xylella fastidiosaTemeculal Dxs (NP_(—)779493); Arabidopsis thaliana Dxs (NP_(—)001078570and/or NP_(—)196699); and homologs thereof. SEQ ID NO:1 represents theParacoccus codon optimized coding sequence for the E. coli dxs gene ofthe present invention. In one aspect, the invention provides nucleicacid molecules and homologs, variants and derivatives of SEQ ID NO:1.The nucleic acid sequences can have 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81-85%, 90-95%, 96-98%, 99%, 99.9% or even higheridentity to SEQ ID NO:1. The present invention provides nucleic acidseach comprising or consisting of a sequence which is a codon optimizedversion of one of the wild-type dxs gene. In another embodiment, theinvention provides nucleic acids each encoding a polypeptide having theamino acid sequence of one of AAC46162, YP 353327, AAV78186,YP_(—)353327, NP_(—)946305, NP_(—)779493, NP_(—)001078570, NP_(—)196699,or homologs thereof having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81-85%, 90-95%, 96-98%, 99%, 99.9% or even higher identitythereto. Exemplary 1-deoxy-D-xylulose-5-phosphate reductoisomerasesinclude E. coli Dxr (BAA32426); Arabidopsis thaliana DXR (AAF73140);Pseudomonas putida KT2440 Dxr (NP_(—)743754 and/or Q88MH4); Streptomycescoelicolor A3(2) Dxr (NP_(—)629822); Rhodobacter sphaeroides 2.4.1 Dxr(YP_(—)352764); Pseudomonas fluorescens PfO-1 Dxr (YP_(—)346389); andhomologs thereof. Exemplary 4-diphosphocytidyl-2C-methyl-D-erythritolsynthases include E. coli IspD (AAF43207); Rhodobacter sphaeroides 2.4.1IspD (YP_(—)352876); Arabidopsis thaliana ISPD (NP_(—)565286); P. putidaKT2440 IspD (NP_(—)743771); and homologs thereof. Exemplary4-diphosphocytidyl-2C-methyl-D-erythritol kinases include E. coli IspE(AAF29530); Rhodobacter sphaeroides 2.4.1 IspE (YP_(—)351828); andhomologs thereof. Exemplary 2C-methyl-D-erythritol 2,4-cyclodiphosphatesynthases include E. coli IspF (AAF44656); Rhodobacter sphaeroides 2.4.1IspF (YP_(—)352877); P. putida KT2440 IspF (NP_(—)743775); and homologsthereof. Exemplary (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthaseinclude E. coli IspG (AAK53460); P. putida KT2440 IspG (NP_(—)743014);Rhodobacter sphaeroides 2.4.1 IspG (YP_(—)353044); and homologs thereof.Exemplary 4-hydroxy-3-methylbut-2-enyl diphosphate reductases include E.coli IspH (AAL38655); P. putida KT2440 IspH (NP_(—)742768); and homologsthereof.

The reactions in the MEV pathway are catalyzed by the following enzymes:acetyl-CoA thiolase, HMG-CoA synthase (E.C. 2.3.3.10), HMG-CoA reductase(E.C. 1.1.1.34), mevalonate kinase (E.C. 2.7.1.36), phosphomevalonatekinase (E.C. 2.7.4.2), mevalonate pyrophosphate decarboxylase (E.C.4.1.1.33), isopentenyl pyrophosphate isomerase (E.C. 5.3.3.2). In oneembodiment, the engineered and/or evolved methylotroph of the presentinvention expresses one or more enzymes from the MEV pathway. Forexample, one or more exogenous proteins can be selected from acetyl-CoAthiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase,phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase andisopentenyl pyrophosphate isomerase. The host organism can also expresstwo or more, three or more, four or more, and the like, including up toall the protein and enzymes that confer the MEV pathway. Exemplaryacetyl-CoA thiolases include NC_(—)000913 REGION: 232413 L.2325315, E.coli; D49362, Paracoccus denitrificans; L20428, S. cerevisiae; andhomologs thereof. Exemplary HMG-CoA synthases include NC_(—)001145complement 19061 . . . 20536, S. cerevisiae; X96617, S. cerevisiae;X83882, A. thaliana; AB037907, Kitasatospora griseola; BT007302, H.sapiens; NC_(—)002758, Locus tag SAV2546, GeneID 1 122571, S. aureus;and homlogs thereof. Exemplary HMG-CoA reductases include NM_(—)206548,D. melanogaster; NC_(—)002758, Locus tag SAV2545, GeneID 1122570, S.aureus; NM_(—)204485, Gallus gallus; AB015627, Streptomyces sp. KO 3988;AF542543, Nicotiana attenuata; AB037907, Kitasatospora griseola;AX128213, providing the sequence encoding a truncated HMGR, S.cerevisiae; NC001145: complement 115734 . . . 1 18898, S. cerevisiae;and homologs thereof. Exemplary mevalonate kinases include L77688, A.thaliana; X55875, S. cerevisiae; and homologs thereof. Exemplaryphosphomevalonate kinases include AF429385, Hevea brasiliensis;NM_(—)006556, H. sapiens; NC_(—)001145 complement 712315 . . . 713670,S. cerevisiae; and homologs thereof. Exemplary mevalonate pyrophosphatedecarboxylase include include X97557, S. cerevisiae; AF290095, E.faecium; U49260, H. sapiens; and homologs thereof. Exemplary isopentenylpyrophosphate isomerases include NP_(—)417365, E. coli Idi; AAC32209,Haematococcus pluvialis Idi; and homologs thereof. SEQ ID NO:2represents the Paracoccus codon optimized coding sequence for the E.coli idi gene of the present invention. In one aspect, the inventionprovides nucleic acid molecules and homologs, variants and derivativesof SEQ ID NO:2. The nucleic acid sequences can have 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81-85%, 90-95%, 96-98%, 99%, 99.9% oreven higher identity to SEQ ID NO:2. The present invention providesnucleic acids each comprising or consisting of a sequence which is acodon optimized version of one of the wild-type idi gene. In anotherembodiment, the invention provides nucleic acids each encoding apolypeptide having the amino acid sequence of one of NP_(—)417365,AAC32209, or homologs thereof having 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81-85%, 90-95%, 96-98%, 99%, 99.9% or even higheridentity thereto.

In some embodiments, the host cell produces IPP via the MEV pathway,either exclusively or in combination with the DXP pathway. In otherembodiments, a host cell's DXP pathway is functionally disabled so thatthe host cell produces IPP exclusively through a heterologouslyintroduced MEV pathway. The DXP pathway can be functionally disabled bydisabling gene expression or inactivating the function of one or more ofthe DXP pathway enzymes.

In some embodiments, the host cell produces IPP via the DXP pathway,either exclusively or in combination with the MEV pathway. In otherembodiments, a host cell's MEV pathway is functionally disabled so thatthe host cell produces IPP exclusively through a heterologouslyintroduced DXP pathway. The MEV pathway can be functionally disabled bydisabling gene expression or inactivating the function of one or more ofthe MEV pathway enzymes.

Provided herein is a method to produce isoprenoids in engineered and/orevolved methylotrophs engineered with the isopentenyl pyrophosphatepathway enzymes. Some examples of isoprenoids include: hemiterpenes(derived from 1 isoprene unit) such as isoprene; monoterpenes (derivedfrom 2 isoprene units) such as myrcene or limonene; sesquiterpenes(derived from 3 isoprene units) such as amorpha-4,11-diene, bisaboleneor farnesene; diterpenes (derived from four isoprene units) such astaxadiene; sesterterpenes (derived from 5 isoprene units); triterpenes(derived from 6 isoprene units) such as squalene; sesquarterpenes(derived from 7 isoprene units); tetraterpenes (derived from 8 isopreneunits) such as f3-carotene or lycopene; and polyterpenes (derived frommore than 8 isoprene units) such as polyisoprene. The production ofisoprenoids is also described in some detail in the published PCTapplications WO2007/139925 and WO/2007/140339.

In another embodiment, the engineered and/or evolved methylotroph of thepresent invention produces isoprene as the carbon-based product ofinterest via the isopentenyl pyrophosphate pathway enzymes and isoprenesynthase (E.C. 4.2.3.27) which converts to dimethylallyl diphosphate toisoprene. Exemplary enzymes include Populus nigra IspS (CAL69918) andhomologs thereof. SEQ ID NO:3 represents the Paracoccus codon optimizedcoding sequence for the P. nigra ispS gene of the present invention. Inone aspect, the invention provides nucleic acid molecules and homologs,variants and derivatives of SEQ ID NO:3. The nucleic acid sequences canhave 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81-85%,90-95%, 96-98%, 99%, 99.9% or even higher identity to SEQ ID NO:3. Thepresent invention provides nucleic acids each comprising or consistingof a sequence which is a codon optimized version of one of the wild-typeispS gene. In another embodiment, the invention provides nucleic acidseach encoding a polypeptide having the amino acid sequence of CAL69918,or homologs thereof having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81-85%, 90-95%, 96-98%, 99%, 99.9° A or even higher identitythereto.

In another embodiment, the engineered and/or evolved methylotroph of thepresent invention produces bisabolene as the carbon-based product ofinterest via the isopentenyl pyrophosphate pathway enzymes andE-alpha-bisabolene synthase (E.C. 4.2.3.38) which converts to farnesyldiphosphate to bisabolene. Exemplary enzymes include Picea abies TPS-bis(AAS47689) and homologs thereof. SEQ ID NO:4 represents the Paracoccuscodon optimized coding sequence for the P. abies tps-bis gene of thepresent invention. In one aspect, the invention provides nucleic acidmolecules and homologs, variants and derivatives of SEQ ID NO:4. Thenucleic acid sequences can have 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81-85%, 90-95%, 96-98%, 99%, 99.9° A or even higheridentity to SEQ ID NO:4. The present invention provides nucleic acidseach comprising or consisting of a sequence which is a codon optimizedversion of one of the wild-type tps-bis gene. In another embodiment, theinvention provides nucleic acids each encoding a polypeptide having theamino acid sequence of AAS47689, or homologs thereof having 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81-85%, 90-95%, 96-98%,99%, 99.9% or even higher identity thereto.

In another embodiment, the engineered and/or evolved methylotroph of thepresent invention produces rubber as the carbon-based product ofinterest via the isopentenyl pyrophosphate pathway enzymes andcis-polyprenylcistransferase (E.C. 2.5.1.20) which converts isopentenylpyrophosphate to rubber. The enzyme cis-polyprenylcistransferase maycome from, for example, Hevea brasiliensis.

In another embodiment, the engineered and/or evolved methylotroph of thepresent invention produce isopentanol as the carbon-based product ofinterest via the isopentenyl pyrophosphate pathway enzymes andisopentanol dikinase.

In another embodiment, the engineered and/or evolved methylotrophproduces squalene as the carbon-based product of interest via theisopentenyl pyrophosphate pathway enzymes, geranyl diphosphate synthase(E.C. 2.5.1.1), farnesyl diphosphate synthase (E.C. 2.5.1.10) andsqualene synthase (E.C. 2.5.1.21). Geranyl diphosphate synthase convertsdimethylallyl pyrophosphate and isopentenyl pyrophosphate to geranyldiphosphate. Farnesyl diphosphate synthase converts geranyl diphosphateand isopentenyl diphosphate to farnesyl diphosphate. A bifunctionalenzyme carries out the conversion of dimethylallyl pyrophosphate and twoisopentenyl pyrophosphate to farnesyl pyrophosphate. Exemplary enzymesinclude Escherichia coli IspA (NP_(—)414955) and homologs thereof.Squalene synthase converts two farnesyl pyrophosphate and NADPH tosqualene. In another embodiment, the engineered and/or evolvedmethylotroph produces lanosterol as the carbon-based product of interestvia the above enzymes, squalene monooxygenase (E.C. 1.14.99.7) andlanosterol synthase (E.C. 5.4.99.7). Squalene monooxygenase convertssqualene, NADPH and O₂ to (S)-squalene-2,3-epoxide. Exemplary enzymesinclude Saccharomyces cerevisiae Erg1 (NP_(—)011691) and homologsthereof. Lanosterol synthase converts (S)-squalene-2,3-epoxide tolanosterol. Exemplary enzymes include Saccharomyces cerevisiae Erg?(NP_(—)011939) and homologs thereof.

In another embodiment, the engineered and/or evolved methylotroph of thepresent invention produces lycopene as the carbon-based product ofinterest via the isopentenyl pyrophosphate pathway enzymes, geranyldiphosphate synthase (E.C. 2.5.1.21, described above), farnesyldiphosphate synthase (E.C. 2.5.1.10, described above), geranylgeranylpyrophosphate synthase (E.C. 2.5.1.29), phytoene synthase (E.C.2.5.1.32), phytoene oxidoreductase (E.C. 1.14.99.n) and ζ-caroteneoxidoreductase (E.C. 1.14.99.30). Geranylgeranyl pyrophosphate synthaseconverts isopentenyl pyrophosphate and farnesyl pyrophosphate to (alltrans)-geranylgeranyl pyrophosphate. Exemplary geranylgeranylpyrophosphate synthases include Synechocystis sp. PCC6803 crtE(NP_(—)440010) and homologs thereof. Phytoene synthase converts 2geranylgeranyl-PP to phytoene. Exemplary enzymes include Synechocystissp. PCC6803 crtB (P37294). Phytoene oxidoreductase converts phytoene, 2NADPH and 2 O₂ to ζ-carotene. Exemplary enzymes include Synechocystissp. PCC6803 crtI and Synechocystis sp. PCC6714 crtI (P21134). ζ-caroteneoxidoreductase converts ζ-carotene, 2 NADPH and 2 O₂ to lycopene.Exemplary enzymes include Synechocystis sp. PCC6803 crtQ-2(NP_(—)441720).

In another embodiment, the engineered and/or evolved methylotroph of thepresent invention produces limonene as the carbon-based product ofinterest via the isopentenyl pyrophosphate pathway enzymes, geranyldiphosphate synthase (E.C. 2.5.1.21, described above) and one of(R)-limonene synthase (E.C. 4.2.3.20) and (4S)-limonene synthase (E.C.4.2.3.16) which convert geranyl diphosphate to a limonene enantiomer.Exemplary (R)-limonene synthases include that from Citrus limon(AAM53946) and homologs thereof. Exemplary (4S)-limonene synthasesinclude that from Mentha spicata (AAC37366) and homologs thereof.

Production of Glycerol or 1,3-Propanediol as the Carbon-Based Productsof Interest

In one aspect, the engineered and/or evolved methylotroph of the presentinvention produces glycerol or 1,3-propanediol as the carbon-basedproducts of interest (FIG. 13). The reactions in the glycerol pathwayare catalyzed by the following enzymes: sn-glycerol-3-P dehydrogenase(E.C. 1.1.1.8 or E.C. 1.1.1.94) and sn-glycerol-3-phosphatase (E.C.3.1.3.21). To produce 1,3,-propanediol, the following enzymes are alsoincluded: sn-glycerol-3-P. glycerol dehydratase (E.C. 4.2.1.30) and1,3-propanediol oxidoreductase (E.C. 1.1.1.202). Exemplarysn-glycerol-3-P dehydrogenases include Saccharomyces cerevisiae darl andhomologs thereof. Exemplary sn-glycerol-3-phosphatases includeSaccharomyces cerevisiae gpp2 and homologs thereof. Exemplarysn-glycerol-3-P. glycerol dehydratases include K. pneumoniae dhaB1-3.Exemplary 1,3-propanediol oxidoreductase include K. pneumoniae dhaT.

Production of 1,4-Butanediol or 1,3-Butadiene as the Carbon-BasedProducts of Interest

In one aspect, the engineered and/or evolved methylotroph of the presentinvention produces 1,4-butanediol or 1,3-butanediene as the carbon-basedproducts of interest. The metabolic reactions in the 1,4-butanediol or1,3-butadiene pathway are catalyzed by the following enzymes:succinyl-CoA dehydrogenase (E.C. 1.2.1.n; e.g., C. kluyveri SucD),4-hydroxybutyrate dehydrogenase (E.C. 1.1.1.2; e.g., Arabidopsisthaliana GHBDH), aldehyde dehydrogenase (E.C. 1.1.1.n; e.g., E. coliA1dH), 1,3-propanediol oxidoreductase (E.C. 1.1.1.202; e.g., K.pneumoniae DhaT), and optionally alcohol dehydratase (E.C. 4.2.1.-).Succinyl-CoA dehydrogenase converts succinyl-CoA and NADPH to succinicsemialdehyde and CoA. 4-hydroxybutyrate dehydrogenase converts succinicsemialdehyde and NADPH to 4-hydroxybutyrate. Aldehyde dehydrogenaseconverts 4-hydroxybutyrate and NADH to 4-hydroxybutanal. 1,3-propanedioloxidoreductase converts 4-hydroxybutanal and NADH to 1,4-butanediol.Alcohol dehydratase converts 1,4-butanediol to 1,3-butadiene.

Production of Polyhydroxybutyrate as the Carbon-Based Products ofInterest

In one aspect, the engineered and/or evolved methylotroph of the presentinvention produces polyhydroxybutyrate as the carbon-based products ofinterest (FIG. 14). The reactions in the polyhydroxybutyrate pathway arecatalyzed by the following enzymes: acetyl-CoA:acetyl-CoAC-acetyltransferase (E.C. 2.3.1.9),(R)-3-hydroxyacyl-CoA:NADP+oxidoreductase (E.C. 1.1.1.36) andpolyhydroxyalkanoate synthase (E.C. 2.3.1.-). Exemplaryacetyl-CoA:acetyl-CoA C-acetyltransferases include Ralstonia eutrophaphaA. Exemplary (R)-3-hydroxyacyl-CoA:NADP+oxidoreductases includeRalstonia eutropha phaB. Exemplary polyhydroxyalkanoate synthase includeRalstonia eutropha phaC. In the event that the host organism also hasthe capacity to degrade polyhydroxybutyrate, the correspondingdegradation enzymes, such as poly[(R)-3-hydroxybutanoate] hydrolase(E.C. 3.1.1.75), may be inactivated. Hosts that lack the ability tonaturally synthesize polyhydroxybutyrate generally also lack thecapacity to degrade it, thus leading to irreversible accumuation ofpolyhydroxybutyrate if the biosynthetic pathway is introduced. Somemethylotrophic bacteria can naturally make poly(3-hydroxybutyrate) orpoly(3-hydroxybutyrate-co-3-hydroxyvalerate), such as Paracoccusdenitrificans [Appl Environ Microbiol, 1996, 62(2):380-384].

Intracellular polyhydroxybutyrate can be measured by solvent extractionand esterification of the polymer from whole cells. Typically,lyophilized biomass is extracted with methanol-chloroform with 10% HClas a catalyst. The chloroform dissolves the polymer, and the methanolesterifies it in the presence of HCl. The resulting mixture is extractedwith water to remove hydrophilic substances and the organic phase isanalyzed by GC.

Production of Lysine as the Carbon-Based Products of Interest

In one aspect, the engineered and/or evolved methylotroph of the presentinvention produces lysine as the carbon-based product of interest. Thereare several known lysine biosynthetic pathways. One lysine biosynthesispathway is depicted in FIG. 15. The reactions in one lysine biosyntheticpathway are catalyzed by the following enzymes: aspartateaminotransferase (E.C. 2.6.1.1; e.g. E. coli AspC), aspartate kinase(E.C. 2.7.2.4; e.g., E. coli LysC), aspartate semialdehyde dehydrogenase(E.C. 1.2.1.11; e.g., E. coli Asd), dihydrodipicolinate synthase (E.C.4.2.1.52; e.g., E. coli DapA), dihydrodipicolinate reductase (E.C.1.3.1.26; e.g., E. coli DapB), tetrahydrodipicolinate succinylase (E.C.2.3.1.117; e.g., E. coli DapD),N-succinyldiaminopimelate-aminotransferase (E.C. 2.6.1.17; e.g., E. coliArgD), N-succinyl-L-diaminopimelate desuccinylase (E.C. 3.5.1.18; e.g.,E. coli DapE), diaminopimelate epimerase (E.C. 5.1.1.7; E. coli DapF),diaminopimelate decarboxylase (E.C. 4.1.1.20; e.g., E. coli LysA). Inone embodiment, the engineered and/or evolved methylotroph of thepresent invention expresses one or more enzymes from a lysinebiosynthetic pathway. For example, one or more exogenous proteins can beselected from aspartate aminotransferase, aspartate kinase, aspartatesemialdehyde dehydrogenase, dihydrodipicolinate synthase,dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase,N-succinyldiaminopimelate-aminotransferase, N-succinyl-L-diaminopimelatedesuccinylase, diaminopimelate epimerase, diaminopimelate decarboxylase,L,L-diaminopimelate aminotransferase (E.C. 2.6.1.83; e.g., Arabidopsisthaliana At4g33680), homocitrate synthase (E.C. 2.3.3.14; e.g.,Saccharomyces cerevisiae LYS21), homoaconitase (E.C. 4.2.1.36; e.g.,Saccharomyces cerevisiae LYS4, LYS3), homoisocitrate dehydrogenase (E.C.1.1.1.87; e.g., Saccharomyces cerevisiae LYS12, LYS11, LYS10),2-aminoadipate transaminase (E.C. 2.6.1.39; e.g., Saccharomycescerevisiae ARO8), 2-aminoadipate reductase (E.C. 1.2.1.31; e.g.,Saccharomyces cerevisiae LYS2, LYS5), aminoadipatesemialdehyde-glutamate reductase (E.C. 1.5.1.10; e.g., Saccharomycescerevisiae LYS9, LYS13), lysine-2-oxoglutarate reductase (E.C. 1.5.1.7;e.g., Saccharomyces cerevisiae LYS1). The host organism can also expresstwo or more, three or more, four or more, and the like, including up toall the protein and enzymes that confer lysine biosynthesis.

Production of Aromatic Compounds as the Carbon-Based Products ofInterest

In certain embodiments, the engineered and/or evolved methylotroph ofthe present invention produces aromatic amino acids, their intermediatesor their derivatives, including but not limited to shikimate,chorismate, prephenate, phenylalanine, tyrosine, tryptophan, orphenylpropranoids, as the carbon-based products of interest. Theengineered and/or evolved methylotroph produces aromatic compounds as anintermediate or product of the methylotrophic or carbon fixation pathwayor as a intermediate or product of host metabolism. In such cases, oneor more transporters may be expressed in the engineered and/or evolvedmethylotroph to export the aromatic compound from the cell. Thesearomatic metabolites can be converted to other products.

In certain embodiments, the engineered and/or evolved methylotroph ofthe present invention produces chorismate as the carbon-based product ofinterested or as a central metabolite precursor to an aromaticcarbon-based product of interest. There are multiple pathways forchorismate biosynthesis. The reactions in one chorismate biosynthesispathway are catalyzed by the following enzymes:2-dehydro-3-deoxyphosphoheptonate aldolase (E.C. 2.5.1.54, e.g., E. coliAroG, AroH, AroF), 3-dehydroquinate synthase (E.C. 4.2.3.4, e.g., E.coli AroB), 3-dehydroquinate dehydratase (E.C. 4.2.1.10, e.g., E. coliAroD), NADPH-dependent shikimate dehydrogenase (E.C. 1.1.1.25, e.g., E.coli AroE), NAD(P)H-dependent shikimate dehydrogenase (E.C. 1.1.1.282,e.g., E. coli YdiB), shikimate kinase (E.C. 2.7.1.71, e.g., E. coli AroLor AroK), 3-phosphoshikimate-1-carboxyvinyltransferase (E.C. 2.5.1.19,e.g., E. coli AroA) and chorismate synthase (E.C. 4.2.3.5, e.g., E. coliAroC). In one embodiment, the engineered and/or evolved methylotroph ofthe present invention expresses one or more enzymes from a chorismatebiosynthetic pathway. For example, one or more exogenous proteins can beselected from 2-dehydro-3-deoxyphosphoheptonate aldolase,3-dehydroquinate synthase, 3-dehydroquinate dehydratase, NADPH-dependentshikimate dehydrogenase, NAD(P)H-dependent shikimate dehydrogenase,shikimate kinase, 3-phosphoshikimate-1-carboxyvinyltransferase andchorismate synthase. The host organism can also express two or more,three or more, four or more, and the like, including up to all theprotein and enzymes that confer chorismate biosynthesis. Chorismateserves as an intermediate to several aromatic compounds includingphenylalanine, tyrosine, tryptophan and the phenylpropranoids.

In certain embodiments, the engineered and/or evolved methylotroph ofthe present invention produces phenylalanine as the carbon-based productof interested or as a precursor to an aromatic carbon-based product ofinterest. There are multiple pathways for phenylalanine biosynthesis.The reactions in one phenylalanine biosynthesis pathway are catalyzed bythe following enzymes: chorismate mutase (E.C. 5.4.99.5, e.g., E. coliPheA or TyrA), prephenate dehydratase (E.C. 4.2.1.51, e.g., E. coliPheA), phenylalanine transaminase (E.C. 2.6.1.57, e.g., E. coli IlvE).In one embodiment, the engineered and/or evolved methylotroph of thepresent invention expresses one or more enzymes from a phenylalaninebiosynthetic pathway. For example, one or more exogenous proteins can beselected from chorismate mutase, prephenate dehydratase andphenylalanine transaminase. The host organism can also express two ormore, three or more, and the like, including up to all the protein andenzymes that confer phenylalanine biosynthesis. Mutants of themethylotrophic Paracoccus denitrificans have been isolated with highaminotransferase (transaminase) activity [Appl Microbiol Biotechnol,1989, 30(3):243-246, DOI: 10.1007/BF00256212].

In certain embodiments, the engineered and/or evolved methylotroph ofthe present invention produces tyrosine as the carbon-based product ofinterested or as a precursor to an aromatic carbon-based product ofinterest. There are multiple pathways for tyrosine biosynthesis. Thereactions in one tyrosine biosynthesis pathway are catalyzed by thefollowing enzymes: chorismate mutase (E.C. 5.4.99.5, e.g., E. coli PheAor TyrA), prephenate dehydrogenase (E.C. 1.3.1.12, e.g., E. coli TyrA),tyrosine aminotransferase (E.C. 2.6.1.57, e.g., E. coli AspC or TyrB).In one embodiment, the engineered and/or evolved methylotroph of thepresent invention expresses one or more enzymes from a tyrosinebiosynthetic pathway. For example, one or more exogenous proteins can beselected from chorismate mutase, prephenate dehydrogeanse and tyrosineaminotransferase. The host organism can also express two or more, threeor more, and the like, including up to all the protein and enzymes thatconfer tyrosine biosynthesis.

Production of γ-Valerolactone as the Carbon-Based Product of Interest

In some embodiments, the engineered and/or evolved methylotroph of thepresent invention is engineered to produce γ-valerolactone as thecarbon-based product of interest. One example γ-valerolactonebiosynthetic pathway is shown in FIG. 16. In one embodiment, theengineered and/or evolved methylotroph is engineered to express one ormore of the following enzymes: propionyl-CoA synthase (E.C. 6.2.1.-,E.C. 4.2.1.- and E.C. 1.3.1.-), beta-ketothiolase (E.C. 2.3.1.16; e.g.,Ralstonia eutropha BktB), acetoacetyl-CoA reductase (E.C. 1.1.1.36;e.g., Ralstonia eutropha PhaB), 3-hydroxybutyryl-CoA dehydratase (E.C.4.2.1.55; e.g., X axonopodis Crt), vinylacetyl-CoA A-isomerase (E.C.5.3.3.3; e.g., C. difficile AbfD), 4-hydroxybutyryl-CoA transferase(E.C. 2.8.3.-; e.g., C. kluyveri OrfZ), 1,4-lactonase (E.C. 3.1.1.25;e.g., that from R. norvegicus). Propionyl-CoA synthase is amulti-functional enzyme that converts 3-hydroxypropionate, ATP and NADPHto propionyl-CoA. Exemplary propionyl-CoA synthases include AAL47820,and homologs thereof. In another embodiment, the invention provides anucleic acid encoding a polypeptide having the amino acid sequence ofSEQ ID NO:5, or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81-85%, 90-95%, 96-98%, 99%, 99.9% or even higheridentity thereto.

Integration of Metabolic Pathways into Host Metabolism

The engineered and/or evolved methylotrophs of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes or proteins participating in one or more carbon productbiosynthetic pathways. Depending on the host methylotroph chosen,nucleic acids for some or all of particular metabolic pathways can beexpressed. For example, if a chosen host methylotroph is deficient inone or more enzymes or proteins for desired metabolic pathways, thenexpressible nucleic acids for the deficient enzyme(s) or protein(s) areintroduced into the host for subsequent exogenous expression.Alternatively, if the chosen host methylotroph exhibits endogenousexpression of some pathway genes, but is deficient in others, then anencoding nucleic acid is needed for the deficient enzyme(s) orprotein(s) to achieve production of desired carbon products from C1compounds. Thus, an engineered and/or evolved methylotroph of theinvention can be produced by introducing exogenous enzyme or proteinactivities to obtain desired metabolic pathways or desired metabolicpathways can be obtained by introducing one or more exogenous enzyme orprotein activities that, together with one or more endogenous enzymes orproteins, produces a desired product such as reduced cofactors, centralmetabolites and/or carbon-based products of interest.

Depending on the metabolic pathway constituents of a selected hostmethylotroph, the engineered and/or evolved methylotrophs of theinvention can include at least one exogenously expressed metabolicpathway-encoding nucleic acid and up to all encoding nucleic acids forone or more energy conversion, carbon fixation, methylotrophic and/orcarbon-based product pathways. For example, a RuMP-derived carbonfixation pathway can be established in a host deficient in a pathwayenzyme or protein through exogenous expression of the correspondingencoding nucleic acid. In a host deficient in all enzymes or proteins ofa metabolic pathway, exogenous expression of all enzyme or proteins inthe pathway can be included, although it is understood that all enzymesor proteins of a pathway can be expressed even if the host contains atleast one of the pathway enzymes or proteins. For example, exogenousexpression of all enzymes or proteins in a carbon fixation pathwayderived from the 3-HPA bicycle can be included, such as the acetyl-CoAcarboxylase, malonyl-CoA reductase, propionyl-CoA synthase,propionyl-CoA carboxylase, methylmalonyl-CoA epimerase,methylmalonyl-CoA mutase, succinyl-CoA:(S)-malate CoA transferase,succinate dehydrogenase, fumarate hydratase,(S)-malyl-CoA/β-methylmalyl-CoA/(S)-citramalyl-CoA lyase,mesaconyl-C1-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase, andmesaconyl-C4-CoA hydratase. Given the teachings and guidance providedherein, those skilled in the art would understand that the number ofencoding nucleic acids to introduce in an expressible form can, atleast, parallel the metabolic pathway deficiencies of the selectedmethylotroph.

Genetic Engineering Methods for Optimization of Metabolic Pathways

In some embodiments, the engineered and/or evolved methylotrophs of theinvention also can include other genetic modifications that facilitateor optimize production of a carbon-based product from C1 compounds orthat confer other useful functions onto the host organism.

In one aspect, the expression levels of the proteins of interest of theenergy conversion pathways, carbon fixation pathways, methylotrophicpathways and/or carbon product biosynthetic pathways can be eitherincreased or decreased by, for example, replacing or altering theexpression control sequences with alternate expression control sequencesencoded by standardized genetic parts. The exogenous standardizedgenetic parts can regulate the expression of either heterologous orendogenous genes of the metabolic pathway. Altered expression of theenzyme or enzymes and/or protein or proteins of a metabolic pathway canoccur, for example, through changing gene position or gene order[Smolke, 2002b], altered gene copy number [Smolke, 2002a], replacementof a endogenous, naturally occurring regulated promoters withconstitutive or inducible synthetic promoters, mutation of the ribosomebinding sites [Wang, 2009], or introduction of RNA secondary structuralelements and/or cleavage sites [Smolke, 2000; Smolke, 2001].

In another aspect, some engineered and/or evolved methylotrophs of thepresent invention may require specific transporters to facilitate uptakeof C1 compounds. In some embodiments, the engineered and/or evolvedmethylotrophs use formate as a C1 compound. If formate uptake islimiting for either growth or production of carbon-based products ofinterest, then expression of one or more formate transporters in theengineered and/or evolved methylotroph of the present invention canalleviate this bottleneck. The formate transporters may be heterologousor endogenous to the host organism. Exemplary formate transportersinclude NP_(—)415424 and NP_(—)416987, and homologs thereof. The presentinvention provides nucleic acids each comprising or consisting of asequence which is a codon optimized version of one of the wild-typeformate transporter genes. In another embodiment, the invention providesnucleic acids each encoding a polypeptide having the amino acid sequenceof one of NP_(—)415424 and NP_(—)416987.

In addition, the invention provides an engineered and/or evolvedmethylotroph comprising a genetic modification conferring to theengineered and/or evolved methylotrophic microorganism an increasedefficiency of using C1 compounds to produce carbon-based products ofinterest relative to the microorganism in the absence of the geneticmodification. The genetic modification comprises one or more genedisruptions, whereby the one or more gene disruptions increase theefficiency of producing carbon-based products of interest from C1compounds. In one aspect, the one or more gene disruptions target genesencoding competing reactions for C1 compounds, reduced cofactors, and/orcentral metabolites. In another aspect, the one or more gene disruptionstarget genes encoding competing reactions for intermediates or productsof the energy conversion, methylotrophic, carbon fixation, and/or carbonproduct biosynthetic pathways of interest. The competing reactionsusually, but not exclusively, arise from metabolism endogenous to thehost cell or organism. Methods for introducing unmarked mutations intothe genome of methylotrophic bacteria such as Paracoccus denitrificanshave been shown previously [J Bacteriol, 1991, 173(21):6962-6970].

A combination of different approaches may be used to identify candidategenetic modifications. Such approaches include, for example,metabolomics (which may be used to identify undesirable products andmetabolic intermediates that accumulate inside the cell), metabolicmodeling and isotopic labeling (for determining the flux throughmetabolic reactions contributing to hydrocarbon production), andconventional genetic techniques (for eliminating or substantiallydisabling unwanted metabolic reactions). For example, metabolic modelingprovides a means to quantify fluxes through the cell's metabolicpathways and determine the effect of elimination of key metabolic steps.In addition, metabolomics and metabolic modeling enable betterunderstanding of the effect of eliminating key metabolic steps onproduction of desired products.

To predict how a particular manipulation of metabolism affects cellularmetabolism and synthesis of the desired product, a theoretical frameworkwas developed to describe the molar fluxes through all of the knownmetabolic pathways of the cell. Several important aspects of thistheoretical framework include: (i) a relatively complete database ofknown pathways, (ii) incorporation of the growth-rate dependence of cellcomposition and energy requirements, (iii) experimental measurements ofthe amino acid composition of proteins and the fatty acid composition ofmembranes at different growth rates and dilution rates and (iv)experimental measurements of side reactions which are known to occur asa result of metabolism manipulation. These new developments allowsignificantly more accurate prediction of fluxes in key metabolicpathways and regulation of enzyme activity [Keasling, 1999a; Keasling,1999b; Martin, 2002; Henry, 2006].

Such types of models have been applied, for example, to analyzemetabolic fluxes in organisms responsible for enhanced biologicalphosphorus removal in wastewater treatment reactors and in filamentousfungi producing polyketides [Pramanik, 1997; Pramanik, 1998a; Pramanik,1998b; Pramanik, 1998c].

In another aspect, some engineered and/or evolved methylotrophs of thepresent invention may require alterations to the pool of intracellularreducing cofactors for efficient growth and/or production of thecarbon-based product of interest from C1 compounds. In some embodiments,the total pool of NAD⁺/NADH in the engineered and/or evolvedmethylotroph is increased or decreased by adjusting the expression levelof nicotinic acid phosphoribosyltransferase (E.C. 2.4.2.11).Over-expression of either the E. coli or Salmonella gene pncB whichencodes nicotinic acid phosphoribosyltransferase has been shown toincrease total NAD⁺/NADH levels in E. coli [Wubbolts, 1990;Berrios-River, 2002; San, 2002]. In another embodiment, the availabilityof intracellular NADPH can be also altered by modifying the engineeredand/or evolved methylotroph to express an NADH:NADPH transhydrogenase[Sauer, 2004; Chin, 2011]. In another embodiment, the total pool ofubiquinone in the engineered and/or evolved methylotroph is increased ordecreased by adjusting the expression level of ubiquinone biosyntheticenzymes, such as p-hydroxybenzoate-polyprenyl pyrophosphate transferaseand polyprenyl pyrophosphate synthetase. Overexpression of thecorresponding E. coli genes ubiA and ispB increased the ubiquinone poolin E. coli [Zhu, 1995]. In the methylotroph Paracoccus denitrificans,p-hydroxybenzoate and mevalonate have been shown to be limiting inproduction of ubiquinone-10 under anaerobic conditions [Appl MicrobiolBiotechnol, 1983, 17(2):85-89, DOI: 10.1007/BF00499856]. In anotherembodiment, the level of the redox cofactor ferredoxin in the engineeredand/or evolved methylotroph can be increased or decreased by changingthe expression control sequences that regulate its expression.

In another aspect, in addition to a C1 compound, some engineered and/orevolved methylotrophs may require a specific nutrients or vitamin(s) forgrowth and/or production of carbon-based products of interest. Forexample, hydroxocobalamin, a vitamer of vitamin B12, is a cofactor forparticular enzymes of the present invention, such as methylmalonyl-CoAmutase (E.C. 5.4.99.2). Required nutrients are generally supplemented tothe growth media during bench scale propagation of such organisms.However, such nutrients can be prohibitively expensive in the context ofindustrial scale bio-processing. In one embodiment of the presentinvention, the host cell is selected from a methylotroph that naturallyproduces the required nutrient(s), such as Protaminobacter ruber orMethylobacterium extorquens, which naturally produces hydroxocobalamin.In an alternate embodiment, the need for a vitamin is obviated bymodifying the engineered and/or evolved methylotroph to express avitamin biosynthesis pathway [Roessner, 1995]. An exemplary biosynthesispathway for hydroxocobalamin comprises the following enzymes:uroporphyrin-III C-methyltransferase (E.C. 2.1.1.107), precorrin-2cobaltochelatase (E.C. 4.99.1.3), cobalt-precorrin-2(C²⁰)-methyltransferase (E.C. 2.1.1.151), cobalt-precorrin-3(C¹⁷)-methyltransferase (E.C. 2.1.1.131), cobalt precorrin-4(C¹¹)-methyltransferase (E.C. 2.1.1.133), cobalt-precorrin 5A hydrolase(E.C. 3.7.1.12), cobalt-precorrin-5B (C¹)-methyltransferase (E.C.2.1.1.195), cobalt-precorrin-6A reductase, cobalt-precorrin-6V(C⁵)-methyltransferase (E.C. 2.1.1.-), cobalt-precorrin-7(C¹⁵)-methyltransferase (decarboxylating) (E.C. 2.1.1.196),cobalt-precorrin-8X methylmutase, cobyrinate A,C-diamide synthase (E.C.6.3.5.11), cob(II)yrinate a,c-diamide reductase (E.C. 1.16.8.1),cob(I)yrinic acid a,c-diamide adenosyltransferase (E.C. 2.5.1.17),adenosyl-cobyrate synthase (E.C. 6.3.5.10), adenosylcobinamide phosphatesynthase (E.C. 6.3.1.10), GTP:adenosylcobinamide-phosphateguanylyltransferase (E.C. 2.7.7.62), nicotinate-nucleotidedimethylbenzimidazole phosphoribosyltransferase (E.C. 2.4.2.21),adenosylcobinamide-GDP:α-ribazole-5-phosphate ribazoletransferase (E.C.2.7.8.26) and adenosylcobalamine-5′-phosphate phosphatase (E.C.3.1.3.73). In addition, to allow for cobalt uptake and incorporationinto vitamin B12, the genes encoding the cobalt transporter areoverexpressed. The exemplary cobalt transporter protein found inSalmonella enterica is overexpressed and is encoded by proteins ABC-typeCo²⁺ transport system, permease component (CbiM, NP_(—)460968), ABC-typecobalt transport system, periplasmic component (CbiN, NP_(—)460967), andABC-type cobalt transport system, permease component (CbiQ,NP_(—)461989).

In some embodiments, the intracellular concentration (e.g., theconcentration of the intermediate in the engineered and/or evolvedmethylotroph) of the metabolic pathway intermediate can be increased tofurther boost the yield of the final product. For example, by increasingthe intracellular amount of a substrate (e.g., a primary substrate) foran enzyme that is active in the metabolic pathway, and the like.

In another aspect, the carbon-based products of interest are or arederived from the intermediates or products of fatty acid biosynthesis.To increase the production of waxes/fatty acid esters, and fattyalcohols, one or more of the enzymes of fatty acid biosynthesis can beover expressed or mutated to reduce feedback inhibition. Additionally,enzymes that metabolize the intermediates to make nonfatty-acid basedproducts (side reactions) can be functionally deleted or attenuated toincrease the flux of carbon through the fatty acid biosynthetic pathwaythereby enhancing the production of carbon-based products of interest.

Growth-Based Selection Methods for Optimization of EngineeredCarbon-Fixing Strains

Selective pressure provides a valuable means for testing and optimizingthe engineered methylotrophs of the present invention. Alternatively, anevolved methylotroph having selected functionality after such selectioncan be further engineered to include additional or alteredfunctionality. In some embodiments, the engineered methylotrophs of theinvention can be evolved under selective pressure to optimize productionof a carbon-based product from a C1 compound or that confer other usefulfunctions onto the host organism. The ability of an optimized engineeredmethylotroph to replicate more rapidly than unmodified counterpartsconfirms the utility of the optimization. Similarly, the ability tosurvive and replicate in media lacking a required nutrient, such asvitamin B12, confirms the successful implementation of a nutrientbiosynthetic module. In some embodiments, the engineered methylotrophscan be cultured in the presence of a limiting amount of C1 compound inorder to select for evolved strains that more efficiently utilize the C1compound. In some embodiments, the engineered methylotrophs of theinvention can be evolved to grow despite the presence of inhibitorycompounds in the C1 feedstock (see, e.g., Example 5).

Evolution can occur as a result of either spontaneous, natural mutationor by addition of mutagenic agents or conditions to live cells. Ifdesired, additional genetic variation can be introduced prior to orduring selective pressure by treatment with mutagens, such asultra-violet light, alkylators [e.g., ethyl methanesulfonate (EMS),methyl methane sulfonate (MMS), diethylsulfate (DES), andnitrosoguanidine (NTG, NG, MMG)], DNA intercalcators (e.g., ethidiumbromide), nitrous acid, base analogs, bromouracil, transposonsm and thelike. Alternatively, genetic variation may be introduced via untargetedgenetic mutagenesis techniques such as transposon insertion.Transposable elements have been used previously to generate phenotypicdiversity in methylotrophic Paracoccus strains [PLoS ONE, 2012,7(2):e32277, DOI: 10.1371/journal.pone.0032277]. The engineeredmethylotrophs can be propagated either in serial batch culture or in aturbidostat as a controlled growth rate.

Alternately or in addition to selective pressure, pathway activity canbe monitored following growth under permissive (i.e., non-selective)conditions by measuring specific product output via various metaboliclabeling studies (including radioactivity), biochemical analyses(Michaelis-Menten), gas chromatography-mass spectrometry (GC/MS), massspectrometry, matrix assisted laser desorption ionization time-of-flightmass spectrometry (MALDI-TOF), capillary electrophoresis (CE), and highpressure liquid chromatography (HPLC).

To generate engineered methylotrophs with improved yield of centralmetabolites and/or carbon-based products of interest, metabolic modelingcan be utilized to guide strain optimization. Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of central metabolites orproducts derived from central metabolites. Modeling can also be used todesign gene knockouts that additionally optimize utilization of theenergy conversion, methylotrophic, carbon fixation and carbon productbiosynthetic pathways. In some embodiments, modeling is used to selectgrowth conditions that create selective pressure towards uptake andutilization of C1 compound(s). An in silico stoichiometric model of hostorganism metabolism and the metabolic pathway(s) of interest can beconstructed (see, for example, a model of the E. coli metabolic network[Edwards, 2002]). The resulting model can be used to compute phenotypicphase planes for the engineered methylotrophs of the present invention.A phenotypic phase plane is a portrait of the accessible growth statesof an engineered methylotroph as a function of imposed substrate uptakerates. A particular engineered methylotroph, at particular uptake ratesfor limiting nutrients, may not grow as well as the phenotypic phaseplane predicts, but no strain should be able to grow better thanindicated by the phenotypic phase plane. Under a variety ofcircumstances, it has been shown the modified E. coli strains evolvetowards, and then along, the phenotypic phase plane, always in thedirection of increasing growth rates [Fong, 2004]. Thus, a phenotypicphase plane can be viewed as a landscape of selective pressure. Strainsin an environment where a given nutrient uptake is positively correlatedwith growth rate are predicted to evolve towards increased nutrientuptake. Conversely, strains in an environment where nutrient uptake areinversely correlated with growth rate are predicted to evolve away fromnutrient uptake.

Fermentation Conditions

The engineered and/or evolved methylotrophs of the present invention arecultured in a medium comprising C1 compound(s) and any requirednutrients. The culture conditions can include, for example, liquidculture procedures as well as fermentation and other large scale cultureprocedures. In one embodiment, the engineered and/or evolvedmethylotroph is grown in a minimal salts medium containing a C1feedstock, such as formate, formic acid, formaldehyde, or methanol. Themedium composition can be optimized for enhanced growth and productionof carbon-based products of interest (see, e.g., Example 1). In oneembodiment, the medium composition is 100 mM sodium bicarbonate, 6 mMsodium chloride, 6 mM sodium nitrate, 11 mM sodium thiosulfate, and 26mM sodium formate in addition to standard MOPS minimal mediumcomponents.

The production and isolation of carbon-based products of interest can beenhanced by employing specific fermentation techniques. One method formaximizing production while reducing costs is increasing the percentageof the carbon that is converted to carbon-based products of interest.During normal cellular lifecycles carbon is used in cellular functionsincluding producing lipids, saccharides, proteins, organic acids, andnucleic acids. Reducing the amount of carbon necessary forgrowth-related activities can increase the efficiency of carbon sourceconversion to output. This can be achieved by first growing engineeredand/or evolved methylotrophs to a desired density, such as a densityachieved at the peak of the log phase of growth. At such a point,replication checkpoint genes can be harnessed to stop the growth ofcells. Specifically, quorum sensing mechanisms [Camilli, 2006; Venturi,2006; Reading, 2006] can be used to activate genes such as p53, p21, orother checkpoint genes. Genes that can be activated to stop cellreplication and growth in E. coli include umuDC genes, theover-expression of which stops the progression from stationary phase toexponential growth [Murli, 2000]. UmuC is a DNA polymerase that cancarry out translesion synthesis over non-coding lesions—the mechanisticbasis of most UV and chemical mutagenesis. The umuDC gene products areused for the process of translesion synthesis and also serve as a DNAdamage checkpoint. UmuDC gene products include UmuC, UmuD, umuD′,UmuD′₂C, UmuD′₂ and UmUD₂. Simultaneously, the carbon productbiosynthetic pathway genes are activated, thus minimizing the need forreplication and maintenance pathways to be used while the carbon-basedproduct of interest is being made.

Alternatively, cell growth and product production can be achievedsimultaneously. In this method, cells are grown in bioreactors with acontinuous supply of inputs and continuous removal of product. Batch,fed-batch, and continuous fermentations are common and well known in theart and examples can be found in [Brock, 1989; Deshpande, 1992].

In one embodiment, the engineered and/or evolved methylotroph isengineered such that the final product is released from the cell. Inembodiments where the final product is released from the cell, acontinuous process can be employed. In this approach, a reactor withorganisms producing desirable products can be assembled in multipleways. In one embodiment, the reactor is operated in bulk continuously,with a portion of media removed and held in a less agitated environmentsuch that an aqueous product can self-separate out with the productremoved and the remainder returned to the fermentation chamber. Inembodiments where the product does not separate into an aqueous phase,media is removed and appropriate separation techniques (e.g.,chromatography, distillation, etc.) are employed.

In an alternate embodiment, the product is not secreted by theengineered and/or evolved methylotrophs. In this embodiment, a batch-fedfermentation approach is employed. In such cases, cells are grown undercontinued exposure to inputs (C1 compounds) as specified above until thereaction chamber is saturated with cells and product. A significantportion to the entirety of the culture is removed, the cells are lysed,and the products are isolated by appropriate separation techniques(e.g., chromatography, distillation, filtration, centrifugation, etc.).

In certain embodiments, the engineered and/or evolved methylotrophs ofthe invention can be sustained, cultured or fermented under anaerobic orsubstantially anaerobic conditions. Briefly, anaerobic conditions refersto an environment devoid of oxygen. Substantially anaerobic conditionsinclude, for example, a culture, batch fermentation or continuousfermentation such that the dissolved oxygen concentration in the mediumremains between 0 and 10% of saturation. Substantially anaerobicconditions also includes growing or resting cells in liquid medium or onsolid agar inside a sealed chamber maintained with an atmosphere of lessthan 1% oxygen. It is highly desirable to maintain anaerobic conditionsin the fermenter to reduce the cost of the overall process.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the C1 feedstockuptake rate by monitoring carbon source depletion over time.

In another embodiment, the engineered and/or evolved methylotrophs canbe cultured in the presence of an electron acceptor, for example,nitrate, in particular under substantially anaerobic conditions. It isunderstood that an appropriate amount of nitrate can be added to aculture to achieve a desired increase in biomass, for example, 1 mM to100 mM nitrate, or lower or higher concentrations, as desired, so longas the amount added provides a sufficient amount of electron acceptorfor the desired increase in biomass. Such amounts include, but are notlimited to, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, asappropriate to achieve a desired increase in biomass. In one embodiment,the engineered and/or evolved methylotroph is a denitrifier that can usenitrate as a terminal electron acceptor and reduce nitrate to nitrogengas. In some embodiments, the engineered and/or evolved methylotroph isderived from methylotrophic denitrifers, such as Paracoccusdenitrificans. Other electron acceptors include fumarate,trimethylammonium oxide, ferricyanide, or dimethyl sulfoxide.

In some embodiments, the engineered and/or evolved methylotrophs of thepresent invention are initially grown in culture conditions with alimiting amount of multi-carbon compounds to facilitate growth. Then,once the supply of organic carbon is exhausted, the engineered and/orevolved methylotrophs transition from heterotrophic to methylotrophicgrowth relying on energy from a C1 compounds in order to producecarbon-based products of interest. The organic carbon can be, forexample, a carbohydrate source. Such sources include, for example,sugars such as glucose, xylose, arabinose, galactose, mannose, fructoseand starch. Other sources of carbohydrate include, for example,renewable feedstocks and biomass. Exemplary types of biomasses that canbe used as feedstocks in the methods of the invention include cellulosicbiomass, hemicellulosic biomass and lignin feedstocks or portions offeedstocks. Such biomass feedstocks contain, for example, carbohydratesubstrates useful as carbon sources such as glucose, xylose, arabinose,galactose, mannose, fructose and starch. Given the teachings andguidance provided herein, those skilled in the art would understand thatrenewable feedstocks and biomass other than those exemplified above alsocan be used for culturing the engineered and/or evolved methylotrophs ofthe invention. In some embodiments, the engineered and/or evolvedmethylotrophs are optimized for a two stage fermentation by regulatingthe expression of the carbon product biosynthetic pathway.

In one aspect, the percentage of input carbon atoms converted tohydrocarbon products is an efficient and inexpensive process. Typicalefficiencies in the literature are ˜<5%. Engineered and/or evolvedmethylotrophs which produce hydrocarbon products can have greater than1, 3, 5, 10, 15, 20, 25, and 30% efficiency. In one example engineeredand/or evolved methylotrophs can exhibit an efficiency of about 10% toabout 25%. In other examples, such microorganisms can exhibit anefficiency of about 25% to about 30%, and in other examples suchengineered and/or evolved methylotrophs can exhibit >30% efficiency.

In some examples where the final product is released from the cell, acontinuous process can be employed. In this approach, a reactor withengineered and/or evolved methylotrophs producing for example, fattyacid derivatives, can be assembled in multiple ways. In one example, aportion of the media is removed and allowed to separate. Fatty acidderivatives are separated from the aqueous layer, which can in turn, bereturned to the fermentation chamber.

In another example, the fermentation chamber can enclose a fermentationthat is undergoing a continuous reduction. In this instance, a stablereductive environment can be created. The electron balance would bemaintained by the release of oxygen. Efforts to augment the NAD/H andNADP/H balance can also facilitate in stabilizing the electron balance.

Consolidated Methylotrophic Fermentation

The above aspect of the invention is an alternative to directlyproducing final carbon-based product of interest as a result ofmethylotrophic metabolism. In this approach, carbon-based products ofinterest would be produced by leveraging other organisms that are moreamenable to making any one particular product while culturing theengineered and/or evolved methylotroph for its carbon source.Consequently, fermentation and production of carbon-based products ofinterest can occur separately from carbon source production in abioreactor.

In one aspect, the methods of producing such carbon-based products ofinterest include two steps. The first-step includes using engineeredand/or evolved methylotrophs to convert C1 compound(s) to centralmetabolites or sugars such as glucose. The second-step is to use thecentral metabolites or sugars as a carbon source for cells that producecarbon-based products of interest. In one embodiment, the two-stageapproach comprises a bioreactor comprising engineered and/or evolvedmethylotrophs; a second reactor comprising cells capable offermentation; wherein the engineered and/or evolved methylotrophsprovides a carbon source such as glucose for cells capable offermentation to produce a carbon-based product of interest. The secondreactor may comprise more than one type of microorganism. The resultingcarbon-based products of interest are subsequently separated and/orcollected.

In some embodiments, the two steps are combined into a single-stepprocess whereby the engineered and/or evolved methylotrophs convert C1compound(s) and directly into central metabolites or sugars such asglucose and such organisms are capable of producing a variety ofcarbon-based products of interest.

The present invention also provides methods and compositions forsustained glucose production in engineered and/or evolved methylotrophswherein these or other organisms that use the sugars are cultured usingC1 compound(s) for use as a carbon source to produce carbon-basedproducts of interest. In such embodiments, the host cells are capable ofsecreting the sugars, such as glucose from within the cell to theculture media in continuous or fed-batch in a bioreactor.

Certain changes in culture conditions of engineered and/or evolvedmethylotrophs for the production of sugars can be optimized for growth.For example, conditions are optimized for C1 compound(s) and theirconcentration(s), electron acceptor(s) and their concentrations,addition of supplements and nutrients. As would be apparent to thoseskilled in the art, the conditions sufficient to achieve optimum growthcan vary depending upon location, climate, and other environmentalfactors, such as the temperature, oxygen concentration and humidity.Other adjustments may be required, for example, an organism's abilityfor carbon uptake.

Advantages of consolidated methylotrophic fermentation include a processwhere there is separation of chemical end products, e.g., glucose,spatial separation between end products (membranes) and time.Additionally, unlike traditional or cellulosic biomass to biofuelsproduction, pretreatment, saccharification and crop plowing areobviated.

The consolidated methylotrophic fermentation process produces continuousproducts. In some embodiments, the process involves direct conversion ofC1 compound(s) to product from engineered front-end organisms to producevarious products without the need to lyse the organisms. For instance,the organisms can utilize 3PGAL to make a desired fermentation product,e.g., ethanol. Such end products can be readily secreted as opposed tointracellular products such as oil and cellulose. In yet otherembodiments, organisms produce sugars, which are secreted into the mediaand such sugars are used during fermentation with the same or differentorganisms or a combination of both.

Processing and Separation of Carbon-Based Products of Interest

The carbon-based products produced by the engineered and/or evolvedmethylotrophs during fermentation can be separated from the fermentationmedia. Known techniques for separating fatty acid derivatives fromaqueous media can be employed. One exemplary separation process providedherein is a two-phase (bi-phasic) separation process. This processinvolves fermenting the genetically-engineered production hosts underconditions sufficient to produce for example, a fatty acid, allowing thefatty acid to collect in an organic phase and separating the organicphase from the aqueous fermentation media. This method can be practicedin both a batch and continuous fermentation setting.

Bi-phasic separation uses the relative immisciblity of fatty acid tofacilitate separation. A skilled artisan would appreciate that bychoosing a fermentation media and the organic phase such that the fattyacid derivative being produced has a high log P value, even at very lowconcentrations the fatty acid can separate into the organic phase in thefermentation vessel.

When producing fatty acids by the methods described herein, suchproducts can be relatively immiscible in the fermentation media, as wellas in the cytoplasm. Therefore, the fatty acid can collect in an organicphase either intracellularly or extracellularly. The collection of theproducts in an organic phase can lessen the impact of the fatty acidderivative on cellular function and allows the production host toproduce more product.

The fatty alcohols, fatty acid esters, waxes, and hydrocarbons producedas described herein allow for the production of homogeneous compoundswith respect to other compounds wherein at least 50%, 60%, 70%, 80%,90%, or 95% of the fatty alcohols, fatty acid esters, waxes andhydrocarbons produced have carbon chain lengths that vary by less than 4carbons, or less than 2 carbons. These compounds can also be produced sothat they have a relatively uniform degree of saturation with respect toother compounds, for example at least 50%, 60%, 70%, 80%, 90%, or 95% ofthe fatty alcohols, fatty acid esters, hydrocarbons and waxes are mono-,di-, or tri-unsaturated.

Detection and Analysis

Generally, the carbon-based products of interest produced using theengineered and/or evolved methylotrophs described herein can be analyzedby any of the standard analytical methods, e.g., gas chromatography(GC), mass spectrometry (MS) gas chromatography-mass spectrometry(GCMS), and liquid chromatography-mass spectrometry (LCMS), highperformance liquid chromatography (HPLC), capillary electrophoresis,Matrix-Assisted Laser Desorption Ionization time-of-flight massspectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR),near-infrared (NIR) spectroscopy, viscometry [Knothe, 1997; Knothe,1999], titration for determining free fatty acids [Komers, 1997],enzymatic methods [Bailer, 1991], physical property-based methods, wetchemical methods, etc.

Sequences Provided by the Invention

Table 4 provides a summary of SEQ ID NOs:1-5 disclosed herein.

TABLE 4 Sequences SEQ ID NO Sequence 1 Codon optimized Escherichia coliDXS gene 2 Codon optimized Escherichia coli IDI gene 3 Codon optimizedPopulus nigra IspS gene 4 Codon optimized Picea abies TPS-bis gene 5Chloroflexus aurantiacus PCS amino acid sequence

EXAMPLES

The examples below are provided herein for illustrative purposes and arenot intended to be restrictive.

Example 1 Optimization of Growth Medium for Paracoccus sp. when UsingFormate as the C1 Compound

Paracoccus zeaxanthinifaciens ATCC 21588, Paracoccus versutus ATCC25364, and Paracoccus denitrificans ATCC 13534 were obtained from theAmerican Type Culture Collection (ATCC).

Strains were tested for the ability to grow aerobically on sodiumformate as a sole source of carbon and/or energy using MOPS minimalmedium (Teknova, Inc.) with sodium formate as a sole carbon source at37C. Unlike previous media used to evaluate the formate-dependent growthof Paracoccus, this medium contains defined levels of trace elementsmolybdenum, boron, copper, zinc, manganese, and other trace metals.

Growth was conducted in various high-throughput machinery capable ofmonitoring growth by light scattering at 600 nm, including a GeminiSpectraNax plate reader (Molecular Devices, Inc.), a Tecan M3000 platereader (Tecan, Inc.), and a BioLector device (m2p-labs, Inc.). For theBioLector, the CO₂ gas content in the culture headspace wascontrollable, as was the humidity.

Paracoccus zeaxanthinifaciens ATCC 21588 was incapable of growth onformate as a sole carbon or energy source. The other two Paracoccusstrains were capable of growth on formate as a sole carbon source, ashas been previously reported [Microbiology, 1979, 114(1):1-13, DOI:10.1099/00221287-114-1-1; Arch Microbiol, 1978, 118(1):21-26, DOI:10.1007/BF00406069].

The effect on growth rate of changes in temperature (T, in degreesCelsius), the partial pressure of CO₂ in the culture headspace gas(pCO₂, in percent by volume), shaking speed (in rpm), and/or theconcentrations (in mM) of sodium formate (HCOONa), sodium nitrate(NaNO₃), sodium thiosulfate (Na₂S₂O₃), sodium chloride (NaCl), andsodium bicarbonate (NAHCO₃) added to the Basal MOPS minimal medium weresystematically evaluated. Combinations examined are shown in Table 2.Paracoccus strains are labelled according to their ATCC number. For eachmeasurement, the instrument used (BioLector; SpectraMax; Tecan), platetype (Flower, Flower Plate; 96 transp, 96-well transparent microtiterplate; 96 opaque, 96-well opaque microtiter plate), use of plate lid,use of humidity control and total culture volume in uL is indicated. N/Aindicates that a particular experimental condition is not applicable forthe instrument used.

TABLE 2 Tested growth conditions for each Paracoccus strain ShakingHumidity ATCC NaHCO₃ NaCl NaNO₃ Na₂S₂O₃ HCOONa T speed Instrument PlateLid Control pCO₂ Vol 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLectorFlower N/A TRUE 5% 1300 25364 150.0 0.0 0.0 0.0 50.0 37.0 1200 BioLectorFlower N/A TRUE 5% 1300 25364 0.0 60.0 0.0 0.0 50.0 37.0 1200 BioLectorFlower N/A TRUE 5% 1300 25364 0.0 0.0 60.0 0.0 50.0 37.0 1200 BioLectorFlower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 80.0 50.0 37.0 1200 BioLectorFlower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 0.0 20.0 37.0 1200 BioLectorFlower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLectorFlower N/A TRUE 5% 1300 13534 150.0 0.0 0.0 0.0 50.0 37.0 1200 BioLectorFlower N/A TRUE 5% 1300 13534 0.0 60.0 0.0 0.0 50.0 37.0 1200 BioLectorFlower N/A TRUE 5% 1300 13534 0.0 0.0 60.0 0.0 50.0 37.0 1200 BioLectorFlower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 80.0 50.0 37.0 1200 BioLectorFlower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 0.0 20.0 37.0 1200 BioLectorFlower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 0.0 50.0 34.0 900 BioLectorFlower N/A TRUE 5% 1300 25364 150.0 0.0 0.0 0.0 50.0 34.0 900 BioLectorFlower N/A TRUE 5% 1300 25364 0.0 60.0 0.0 0.0 50.0 34.0 900 BioLectorFlower N/A TRUE 5% 1300 25364 0.0 0.0 60.0 0.0 50.0 34.0 900 BioLectorFlower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 80.0 50.0 34.0 900 BioLectorFlower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 0.0 20.0 34.0 900 BioLectorFlower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 0.0 50.0 34.0 900 BioLectorFlower N/A TRUE 5% 1300 13534 150.0 0.0 0.0 0.0 50.0 34.0 900 BioLectorFlower N/A TRUE 5% 1300 13534 0.0 60.0 0.0 0.0 50.0 34.0 900 BioLectorFlower N/A TRUE 5% 1300 13534 0.0 0.0 60.0 0.0 50.0 34.0 900 BioLectorFlower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 80.0 50.0 34.0 900 BioLectorFlower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 0.0 20.0 34.0 900 BioLectorFlower N/A TRUE 5% 1300 25364 10.7 4.3 4.3 40.0 45.7 36.6 1157 BioLectorFlower N/A TRUE 5% 1300 13534 75.0 4.3 4.3 5.7 45.7 36.6 1157 BioLectorFlower N/A TRUE 5% 1300 13534 10.7 4.3 30.0 5.7 45.7 36.6 1157 BioLectorFlower N/A TRUE 5% 1300 25364 42.9 17.1 17.1 0.0 32.9 35.3 1029BioLector Flower N/A TRUE 5% 1300 13534 0.0 17.1 17.1 22.9 32.9 35.31029 BioLector Flower N/A TRUE 5% 1300 13534 42.9 17.1 0.0 22.9 32.935.3 1029 BioLector Flower N/A TRUE 5% 1300 25364 101.0 6.1 6.1 11.426.7 34.4 937 BioLector Flower N/A TRUE 5% 1300 25364 101.0 6.1 6.1 0.026.7 34.4 937 BioLector Flower N/A TRUE 5% 1300 13534 122.4 29.4 14.716.3 67.8 34.4 937 BioLector Flower N/A TRUE 5% 1300 13534 122.4 29.414.7 0.0 67.8 34.4 937 BioLector Flower N/A TRUE 5% 1300 21588 0.0 0.00.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 25364 0.0 0.00.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 13534 0.0 0.00.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 21588 0.0 0.00.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 25364 0.0 0.00.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 13534 0.0 0.00.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 21588 0.0 0.00.0 0.0 50.0 31.0 N/A SpectraMax 96 transp TRUE N/A N/A 150 25364 0.00.0 0.0 0.0 50.0 31.0 N/A SpectraMax 96 transp TRUE N/A N/A 150 135340.0 0.0 0.0 0.0 50.0 31.0 N/A SpectraMax 96 transp TRUE N/A N/A 15021588 0.0 0.0 0.0 0.0 50.0 31.0 Linear (8.5) Tecan 96 transp TRUE N/AN/A 150 25364 0.0 0.0 0.0 0.0 50.0 31.0 Linear (8.5) Tecan 96 transpTRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 31.0 Linear (8.5) Tecan 96transp TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5)Tecan 96 opaque TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 Linear(8.5) Tecan 96 opaque TRUE N/A N/A 100 13534 0.0 0.0 0.0 0.0 50.0 37.0Linear (8.5) Tecan 96 opaque TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.037.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 100 25364 0.0 0.0 0.0 0.050.0 37.0 1200 BioLector 96 opaque TRUE FALSE FALSE 150 25364 0.0 0.00.0 0.0 50.0 37.0 1200 BioLector 96 opaque TRUE FALSE FALSE 100 135340.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque TRUE FALSE FALSE 15013534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque TRUE FALSEFALSE 100 25364 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaqueTRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96opaque TRUE N/A N/A 100 13534 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5)Tecan 96 opaque TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 37.0 Linear(8.5) Tecan 96 opaque TRUE N/A N/A 100 25364 0.0 0.0 0.0 0.0 50.0 37.0800 BioLector 96 opaque TRUE TRUE FALSE 150 25364 0.0 0.0 0.0 0.0 50.037.0 800 BioLector 96 opaque TRUE TRUE FALSE 100 13534 0.0 0.0 0.0 0.050.0 37.0 800 BioLector 96 opaque TRUE TRUE FALSE 150 13534 0.0 0.0 0.00.0 50.0 37.0 800 BioLector 96 opaque TRUE TRUE FALSE 100 25364 0.0 0.00.0 0.0 50.0 37.0 1200 BioLector 96 opaque FALSE TRUE FALSE 150 253640.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque FALSE TRUE FALSE 10013534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque FALSE TRUEFALSE 150 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque FALSETRUE FALSE 100 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/ATRUE FALSE 1300 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector FlowerN/A TRUE FALSE 1300

The particular values for salt, bicarbonate, formate, thiosulfate, ornitrate concentration, as well as temperature, were chosen byimplementing a Nelder-Mead simplex optimization algorithm (as describedin Chapter 18 of Chemometrics: a textbook ISBN: 0444426604) using thestarting simplices with points chosen from the following possibilities:temperature, 34° C. or 37° C.; sodium bicarbonate, 150 mM or 0 mM;sodium chloride, 60 mM or 0 mM; sodium formate, 20 mM or 50 mM; sodiumnitrate, 60 mM or 0 mM; sodium thiosulfate, 0 mM or 80 mM; shakingspeed, 1200 rpm or 900 rpm. Growth was evaluated for both 25364 and13534 at each chosen medium condition. For each strain and mediumcondition, a score indicative of the growth was calculated as the time(in hours) to 50% of the maximum growth attained in the entireexperiment minus the time to 5% of the maximum growth. This metric iseasy to compute and avoids penalizing conditions with longer lag phases.

From the scores, new medium conditions were calculated according to theNelder-Mead simplex algorithm. These medium conditions were tested aswell. The growth under the new condition as well as the old ones wasused to define the points of a new simplex, and the process repeated.

After several rounds of the medium optimization process, a satisfactorymedium condition, allowing for faster growth than the initially chosenmedium conditions, was obtained. In total, 68 different uniquemedium/strain/temperature/shaking conditions were examined. The fastestgrowth on formate as a sole carbon source was obtained for ATCC strain25364. Under the optimal conditions, the medium consisted of 100 mMsodium bicarbonate, 6 mM sodium chloride, 6 mM sodium nitrate, 11 mMsodium thio sulfate, and 26 mM sodium formate in addition to standardMOPS minimal medium components. The optimal growth temperature was 34°C. Under these conditions, ATCC strain 25364 had a growth rate of >0.7hr⁻¹, corresponding to a doubling time of 0.95 hr.

ATCC strain 25364 was found capable of growth at rates in excess of 0.4hr⁻¹ using a simpler medium composition of MOPS minimal medium plus 50mM sodium formate.

Large concentrations of thiosulfate were found to give slowed, biphasicgrowth curves for both strains of Paracoccus tested. In general,moderate concentrations of sodium nitrate showed improved growth. Growthat 34° C. was slightly better than growth at 37° C., and both of thesetemperatures were significantly better than growth at 30° C.

Example 2 Automatable Protocol for Conjugative Transfer of Plasmids fromE. coli Donors to Paracoccus sp

E. coli strain 517-1 was obtained from the Yale E. coli Genetic StockCenter. Paracoccus denarificans PD1222 was obtained from Stephen Spiro(University of Texas at Dallas). E. coli S17-1 strain is tra+, meaningit is able to mobilize for conjugative transfer those plasmids harboringa mob+genotype. Plasmid pDIY313K, obtained from Dariusz Bartosik(University of Warsaw, Poland) and described by his laboratory [JMicrobiol Methods, 2011, 86(2):166-74, DOI:10.1016/j.mimet.2011.04.016], and introduced into E. coli 517-1 bystandard methods.

E. coli S17-1 was grown on Luria broth with carbenicillin overnight.Paracoccus versutus ATCC 25364 was grown overnight on MOPS minimalmedium (Teknova, Inc.) with 50 mM sucrose and 40 mM sodium nitrate.

The next day, E. coli S17-1 was subcultured in antibiotic-free Luriabroth for >4 hr. Paracoccus strains were subcultured in identicalMOPS/sucrose/nitrate medium. After cultures of both E. coli andParacoccus had reach log phase, with optimal density greater than 1.0cm⁻¹, cultures were mixed in equal volumes in wells of a standard,SBS-format 96-well plate. No effort was made to pellet the cells, toimmobilize cells on porous filters, to culture the cells on solid media,or to otherwise manipulate the mixtures. Cultures were simply mixed inequal volumes and incubated overnight at 37° C. without agitation.

After overnight incubation, the mixed cultures were diluted in PBS anddilutions were plated on MOPS/sucrose/kanamycin agar. E. coli cannot usesucrose as a carbon source, and only strains carrying pDIY313K can growin the presence of kanamycin. Thus on these plates onlytransconjugants—strains of Paracoccus containing plasmid DNA andexpressing plasmid-derived kanamycin resistance genes—can grow. Inparallel we plated the same dilutions on MOPS/sucrose agar withoutkanamycin, in order to calculate the cell concentration of totalParacoccus cells used in the experiment and to calculate thetransconjugation frequency (colonies of plasmid-bearing Paracoccusisolated per colony of recipient Paracoccus cell).

Using this simple technique we were able to demonstrate conjugationfrequencies of 10⁻⁵ using Paracoccus denitrificans PD1222 and 2×10⁻⁷using Paracoccus versutus. It should be emphasized that this frequencywas determined via a protocol which did not require non-selective growthon soft medium, the use of filters, or any centrifugation steps. Thesesteps are required in protocols for conjugation frequently taught in theliterature. For example, Bartosik [J Microbiol Methods, 2011,86(2):166-74, DOI: 10.1016/j.mimet.2011.04.016] teaches that cells mustbe grown, pelleted by centrifugation, washed, resuspendend, mixed,immobilized on a porous filter, grown under non-selective agarovernight, removed from the filter by washing, pelleted, and finallyplated on selective medium. The lack of any such laborious cellmanipulation procedures in our protocol is essential for conduction ofthe protocol on a robotics-based liquid-handling platform, wherecentrifugation and resuspension operations are much more error-prone,hard to implement, and/or unreliable in comparison with simple liquidhandling steps.

Example 3 Genome Sequencing of Paracoccus Strains

Genomic DNA was isolated from Paracoccus zeaxanthinifaciens ATCC 21588,Paracoccus versutus ATCC 25364, and Paracoccus denitrificans ATCC 13534using a Wizard Genomic DNA Isolation Kit (Promega, Inc.). The resultingDNA samples were fragmented and converted to paired-end libraries forwhole-genome shotgun sequencing on a 454 pyrosequencing platform (Roche,Inc.).

For Paracoccus denitrificans, 37,585,886 paired reads, each 100 nt inlength, were obtained. This represents approximately 3.7 gigabases ofsequence data, or approximately 730-fold coverage of the 5.2 megabasegenome of Paracoccus denitrificans PD1222 (Genbank accession numbersCP000489, CP000490, and CP000491 for chromosome 1, chromosome 2, and a653,815 bp megaplasmid, respectively). Reads were assembled first byde-novo assembly and second by mapping the de-novo contigs to thepublished PD1222 genome.

The resulting reads could be assembled into a crude whole-genomeassembly of 351 scaffolds comprising 21,972,742 total reads. The maximumscaffold was 7974 nt and minimum-length scaffold 2004 nt.

Example 4 Analysis of Methylerythritol Pathway in Paracoccus

The Paracoccus denitrificans PD1222 genome has been published. Throughmanual inspection and BLAST searching we found homologs to all but onemember of the methylerythritol pathway for isoprenoid biosynthesis. TheParaoccus gene homologs are shown in Table 3. Gene names refer tostandard names given to E. coli genes (see accession Genbank accessionU000096 for more information). Names for Paracoccus genes correspond tothe nomenclature annotated as part of the Paracoccus PD 1222 genomesequence, available at Genbank accession numbers CP000489 for chromosome1 and CP000490 for chromosome 2.

TABLE 3 Methylerythritol pathway gene homologs in Paracoccusdenitrificans PD122 P. denitrificans PD1222 E. coli gene PD1222 genechromosome # dxs Pden_0400 1 dxr Pden_3997 2 ispD Pden_3667 (KEGG); none(Metacyc) 2 ispE Pden_0423 1 ispF Pden_3667 2 ispG Pden_1820 1 ispHPden_3619 2 idi no type I or type II homologues ? ispA Pden_0399 1

The sole member of the methylerythritol pathway missing a homolog fromthe P. denitrificans PD1222 genome is the gene idi, encodingisopentenyl-diphosphate Δ-isomerase (E.C. 5.3.3.2). It is responsiblefor interconverting isopentenyl diphosphate (IPPP) and dimethylallyldiphosphate (DMAP). However, several studies have shown that this geneis not required for pathway activity, since the preceding pathway step,coded for in E. coli by the ispH gene that shares homology withpredicted Paracoccus gene Pden_(—)3619, generates both IPPP and DMAP tosome degree [Lipids, 2008, 43(12):1095-1107, DOI:10.1007/s11745-008-3261-7].

P. denitrificans is known to contain prenylated quinones as constituentsof its cell membrane [Biochem Eng J, 2003, 16(2):183-190, DOI:10.1016/51369-703X(03)00035-4]. These compounds are indicative ofterpene production. The presence of gene homologs for themethylerythritol pathway indicate that this pathway is responsible forformation of terpenoids in this organism.

Example 5 Selection for Populations of Paracoccus Versutus with ImprovedDoubling Times on Electrolytically Generated Formate

A computer-controlled continuous culture device that can be operated asa chemostat or a turbidostat was constructed. The device has a workingvolume between 20 and 50 mL (not yet tested above 50 mL). The deviceuses air pressure to move liquids throughout the fluidics system, and anarray of solenoid valves to direct fluid flow. The culture is mixed andaerated by the turbulence created by sparging with air at a flow rateof >10 vvm. The valves are controlled by an Arduino Mega 2560microprocessor. The Arduino also interfaces with the sensors and controlmechanisms of the device. The optical density (OD) of the culture isdetermined by an infrared LED-photodiode pair which measures thetransmittance of light across the culture vessel.

From Edward Rode at DNV, Inc. (formerly Det Norske Veritas), we obtainedtwo samples of formate generated by DNV from electricity and carbondioxide by electrolysis. The first solution received from DNV contained0.5 M potassium chloride (electrolyte), 0.5 M potassium formate, and 2.5M sodium bicarbonate. The second solution as received from DNV containedapproximately 0.5 M potassium chloride (electrolyte), and 0.56 Mpotassium formate. This solution was directly obtained from the cathodicchamber of DNV's electrolysis reactor without any upgrading orpurifying, and thus it may contain other uncharacterized contaminants orother agents which inhibit the growth of bacteria. These may arise frommetals or plastics leaching into solution, from uncharacterizedelectrochemical reactions going on in parallel with the cathodicreduction of bicarbonate (i.e. dissolved CO₂) to formate salts, or fromother processes. The ability for engineered cells to operate directly onsuch solutions would be of interest for the development of low-costelectricity-to-chemicals bioconversion processes.

We verified that electrolytically generated formate reduces the growthrate of Paracoccus versutus. Overnight cultures of MOPS minimal formatemedium were inoculated into MOPS minimal medium containing eithervarious dilutions of sodium formate (Sigma-Aldrich) or various dilutionsof formate sourced from DNV's first sample. Growth was uninhibited bypure sodium formate at the highest concentration tested, 50 mM. Incontrast growth was strongly inhibited by electrolytically generatedformate, with no growth observed above 20 mM formate concentration, andonly weak growth at concentrations above 10 mM. However at lowerconcentrations, electrolytic formate supported Paracoccus growth atrates similar to pure sodium formate.

In an effort to select for strains with an increased ability to thriveon electrolytically generated formate, Paracoccus versutus wasinoculated into the culture device. MOPS minimal medium with commercial(Sigma-Aldrich) sodium formate at 50 mM flowed through the device atflow rates controlled by the Arduino in order to maintain with a targetOD setpoint between 0.2 and 0.3 cm⁻¹. In practice this flow rate wasbetween 6 to 9 mL hr⁻¹. The working volume of the device was 24 mL,meaning the dilution rate was between 0.25 hr⁻¹ and 0.3 hr⁻¹.Periodically throughout the continuous culture, samples of the culturewere taken and preserved as a glycerol stock at −80° C.

After 48 generations of growth, the medium feed was changed to be amixture of 75 volume % MOPS minimal medium with 50 mM sodium formate,and 25 volume % MOPS minimal medium with 50 mM electrolytic formate(from DNV's second sample). The culture was incubated continuously for32 more generations of growth.

After the conclusion of the experiment, glycerol stocks previously takenfrom the reactor population and reserved at −80° C. were revived andcultured in MOPS formate minimal medium to determine if any improvementsin growth on formate had taken place. We found that the P. versutuspopulation sampled from the turbidostat zero generations of growth hadmuch lower growth rates on 50 mM electrolytic formate (MOPS-EF) mediumthan on 50 mM pure sodium formate (MOPS-PF) medium (0.32 hr⁻¹ forMOPS-PF vs. 0.26 hr⁻¹ for MOPS-EF). Populations sampled from the reactorat later times had faster growth rates, as shown in Table 4. Clones fromthis population can be used as hosts for production of fuels or othercarbon products of interest because of their ability to better toleratesolutions of electrolytic formate as their sole source of carbon andenergy, and their ability to grow more quickly than wild-type P.versutus under these conditions.

TABLE 4 Growth rates of evolved Paraoccus strains Generations ofGenerations of Selection on MOPS-EF Growth MOPS-PF Growth Selection,total MOPS-EF Rate, hr⁻¹ rate, hr⁻¹ 0 0 0.25 0.32 35 0 0.32 0.43 80 320.41 0.41

Example 6 High Intensity Bioreactor Cultivation of Paracoccus on FormateSalts

To our knowledge, the high-cell density bioreactor cultivation ofindustrially relevant, genetically tractable microbes using formate asthe sole source of carbon and energy has not been reported. We sought todemonstrate the high-cell density bioreactor cultivation of Paracoccusversutus under process-relevant conditions.

In a series of two experiments comprising 12 different fed-batch runs,the reactors were initially charged with 0.5 L of a formate minimalmedium based on the recipe of R minimal medium, but with emendations ofsodium molybdate, sodium selenite, thiamin, and cobalamin. The reactorswere inoculated with overnight flask cultures of Paracoccus versutus.After the initial charge of formate in the reactor was consumed,supplemental feeding was begun by flowing 8.0 M ammonium formate to thereactors. Over the course of 12 experiments, we studied the effect ofinitial inoculum size, feeding rate, and aerobicity on rates of formateconsumption and of biomass and CO₂ formation. We monitored formateconsumption by HPLC, CO₂ emission through IR-based off-gas measurement,and biomass formation by total insoluble solids.

Initially, initial biomass concentration corresponding to OD 0.1 wasused; however, biomass measurement by weight was unreliable due tomineral precipitation during fermentation. Subsequently, initial biomassconcentration was OD1.0 and we used a dilute-acid wash during theprocessing of biomass samples in order to remove inorganic precipitates.We assumed that biomass contained 0.5 g-C g⁻¹. This assumption allowedus to close carbon balances around the aerobic, high-inoculum runs towithin 10%, indicating that the assumption was reasonable andconstituting a consistency check on our HPLC and off-gas measurements.

FIG. 17 depicts sample fermentation data for an aerobic fermentationfeed of 10 mM hr⁻¹. From the beginning of the formate feed at 3.45 hrpost-inoculation to near the end of the run at 50 hr, the formate levelremained below detection limits. The 75 mmol-C of biomass formedresulted in a final biomass concentration of 2.6 g L⁻¹, implying thatspecific formate consumption rates were at or above 0.51 g formategDCW⁻¹ hr⁻¹ throughout the fermentation. In this run, 91% of the formateconsumed was converted to CO₂ that left the reactor and 8.9% wasaccounted for by biomass formation. The total of biomass formation andCO₂ emisson accounted for 100.0% of the carbon used, a figure whichvaried between 100 and 103% across other runs. These rates correspond toa specific carbon fixation flux of 8 mmol C gDCW⁻¹ hr⁻¹ or a volumetriccarbon fixation flux of >1.68 mmol L⁻¹ hr⁻¹.

Nominal feed rates of 10, 30, and 100 mM hr⁻¹ were studied for aerobicfermentations. Only the 100 mM hr⁻¹ condition showed any evidence offormate accumulation, although this feed was only tried with alow-inoculum size condition (OD0.1). The 10 and 30 mM hr⁻¹ feed ratesdid not show any formate accumulation until the end of thefermentations, indicating that the capacity of the culture for formateutilization was greater than 30 mM hr⁻¹ under the densities used forcultivation.

Respiration of formic acid or formate is a proton-consuming, i.e. pHincreasing process. In these experiments, the medium pH was heldconstant at 7.0 by the addition of concentrated phosphoric acid.Ammonium formate was chosen as a formate source because it provides anadditional means of pH control (ammonium formate solutions are neutralin pH) and because it was hoped that as formate was consumed, ammoniumwould not accumulate due to the potential for offgassing of ammonia. Wemeasured ammonium accumulation in the reactors by ion chromatography. AtpH 7.0, ammonium offgassing did not occur to an appreciable extent,because 86-102% of the fed ammonium formate accumulated as ammonium inthe medium Ammonium accumulation likely limited the end-point biomasstiter attained in most of the fermentations, as it accumulated tosupra-molar concentrations in many of the vessels.

Paracoccus versutus can grow anaerobically using nitrate as an electronacceptor. We carried out nitrate-based anaerobic formate bioconversionusing feeds that contained 8.0 M ammonium formate and 3.1 M sodiumnitrate. When feeding was begun, the reactors were brought underanaerobiosis by sparging with nitrogen. Anaerobic fermentations alsoconsumed formate at high rates, up to 0.67 g L⁻¹ hr⁻¹ in the experimentsdescribed here. Maximal biomass attained under anaerobic conditions was1.3 gDCW L⁻¹. The low nitrogen sparging flow rates were incompatiblewith CO₂ measurement in the off-gas, so carbon balances are notavailable.

Initial results indicated that nitrate was converted to nitrite and thatnitrite accumulated stoichiometrically in the reactor and was notfurther reduced. We successfully eliminated nitrite accumulation in thereactor by doubling the amount of copper in the medium and reducing thelevel of nitrate from 3.1 M to 3.0 M. These results demonstrate thatParacoccus is capable of anaerobic formate consumption with completenitrate respiration to dinitrogen gas Ammonium, formate and nitratereached levels of 1100, 1500 and 540 mmol L⁻¹, respectively.

Example 7 Computing Mass Transfer Limitations of Synthesis Gas VersusFormate as a Feedstock

The mass tranfer limitations of synthesis gas (composed of molecularhydrogen and carbon monoxide) from the gas to liquid phase isillustrated here. For the purpose of this analysis, an ideal engineeredorganism that has an unlimited capacity to (i) metabolize dissolvedaqueous-phase synthesis gas and (ii) convert it to a desired fuel at100% of the theoretical yield is assumed. Under these conditions, therate of fuel production per unit of reactor volume can depend solely onthe rate at which synthesis gas can be transferred from the gas phase tothe liquid phase.

Fuel productivity Pin units of g·L⁻¹h⁻¹ can be expressed as the productof fuel molecular weight m_(F), fuel molar yield on synthesis gasY_(F/S), the biomass concentration in a bioreactor X, and the specificcellular uptake rate of synthesis gas q_(S), as shown in the equationbelow.

P=M _(F) Y _(F/S) Xq _(S)

At steady state, the bulk hydrogen uptake rate Xq_(S) is equal to therate of synthesis gas transfer from gas to liquid, meaning theproductivity can be expressed as in the equation below, where C* is theliquid-phase solubility of synthesis gas, C_(L) is the liquid-phaseconcentration of synthesis gas, and K_(L)a is the mass transfercoefficient for synthesis gas transport from the gas phase (e.g., asbubbles sparged into the reactor) to the liquid. K_(L)a is a complexfunction of reactor geometry, bubble size, superficial gas velocity,impeller speed, etc. and is best regarded as an empirical parameter thatneeds to be determined for a given bioreactor setup.

P=m _(F) Y _(F/S) K _(L) a(C*−C _(L))

Again, as a best-case scenario, an ideal engineered organism capable ofmaintaining rapid synthesis gas uptake rates even at vanishingly lowsynthesis gas concentrations (i.e. that q_(S) is not a function of C_(L)even as C_(L) tends to zero) is assumed. This assumption maximizes thefuel productivity at P=m_(F)Y_(F/S)K_(L)aC*.

For a fixed production target t, say 0.5 t d⁻¹ (equivalent to 20800 gh⁻¹), the productivity P determines the required reactor volume Vbecause V=t/P. Thus, both fuel productivity and reactor volumes, evenassuming “perfect” organisms, are bounded by achieveable K_(L)a values,as shown in the equations below.

P = (m_(F)Y_(F/S)C^(*))K_(L)a$V = \frac{t}{\left( {m_{F}Y_{F/S}C^{*}} \right)K_{L}a}$

Maximal productivity corresponds to minimal reaction volumes, and occursat maximal values of M_(F)Y_(F/S)C*K_(L)a. The fuel yield cannot exceedthe stoichiometric maximal yield. For the fuel isooctanol, thestoichiometric maximal yield is determined from the balanced chemicalequation 8 CO+16 H₂→C₈H₁₈O+7 H₂O, which shows that 16 moles of H₂ and 8moles of CO are required for each mole of isooctanol produced. Atatmospheric pressure, C* is unlikely to greatly exceed 0.75 mM, thesolubility of H₂ in pure water (CO has approximately the same solubilityas H₂). Using these representative values for representative values form_(F), Y_(F/S), C* and t, the relationships between K_(L)a and P as wellas between K_(L)a and t are shown (FIG. 18).

Alternative electron donors have the potential to solve both the safetyproblem and the mass transfer problem presented by hydrogen. An idealnon-synthesis gas vector for carrying electrical energy would have (a) ahighly negative standard reduction potential and (b) establishedhigh-efficiency technology to for converting electricity into thevector. Unlike synthesis gas, however, it would (c) have a lowpropensity to explode when mixed with air, and (d) have high watersolubility under bio-compatible conditions. Formic acid, HCOOH, or itssalts, satisfies these conditions. Formic acid is stoichiometricallyequivalent to H₂+CO₂, and formate has as standard reduction potentialnearly identical to that of hydrogen. Since both formic acid and formatesalts are highly soluble in water, the mass transfer limitationsdiscussed above for hydrogen do not apply. However, a modified form ofthe fuel productivity equation, written for formic acid (A) instead ofhydrogen (H), still applies, as shown below.

P=m _(F) Y _(F/A) Xq _(A)

Unlike hydrogen-powered electrofuels bioproduction, limits onformate-powered fuel productivity P stem only from the attainable yield,the biomass concentration in the reactor, and the specific uptake rate.We assume Y_(F/A), the molar yield of fuel on formic acid, is thestoichiometric maximum, whose value is the same as for hydrogen, 0.0467mol isooctanol (mol HCOOH)⁻¹. For high-cell density cultivations of E.coli, biomass concentrations of X=50 gDCW L⁻¹ are attainable, althoughthese values have not been observed for growth on formate or in minimalmedium. For Paracoccus versutus, naturally capable of growing onformate, observed values of were 0.0368 mol formate. gDCW⁻¹h⁻¹ [Kelly,1979]. The representative values for q_(A) and X imply a maximalisooctanol productivity on formate of about 10 g·L⁻¹h⁻¹.

On the γ-axis of FIG. 18, the range of reported K_(L)a attainable inlarge-scale stirred-tank bioreactors is shown. Although there are manyreports of higher K_(L)a values in laboratory-scale reactors, duringscale up the inevitable increase in volume-to-surface area ratios meansthat maintaining high K_(L)a values is for practical purposesimpossible. The maximum of the indicated range of 10-800 h⁻¹ translatesto a best-case productivity of 4 g·L⁻¹·h⁻¹, which implies a best-casereactor volume of 6,400 L. The best-case productivity on formate is 10g·L⁻¹h⁻¹, implying a reactor volume less than half as large would berequired to achieve the same production. Most sources that give K_(L)avalues for large scale reactors have values much closer to 100 h⁻¹,meaning the best-case productivity using formate as the energy sourcewould be more than 15 times larger than on synthesis gas.

Example 8 Engineered Organisms Producing Butanol

The enzyme beta-ketothiolase (R. eutropha PhaA or E. coli AtoB) (E.C.2.3.1.16) converts 2 acetyl-CoA to acetoacetyl-CoA and CoA.Acetoacetyl-CoA reductase (R. eutropha PhaB) (E.C. 1.1.1.36) generatesR-3-hydroxybutyryl-CoA from acetoacetyl-CoA and NADPH. Alternatively,3-hydroxybutyryl-CoA dehydrogenase (C. acetobutylicum Hbd) (E.C.1.1.1.30) generates S-3-hydroxybutyryl-CoA from acetoacetyl-CoA andNADH. Enoyl-CoA hydratase (E. coli MaoC or C. acetobutylicum Crt) (E.C.4.2.1.17) generates crotonyl-CoA from 3-hydroxybutyryl-CoA. Butyryl-CoAdehydrogenase (C. acetobutylicum Bcd) (E.C. 1.3.99.2) generatesbutyryl-CoA and NAD(P)H from crotonyl-CoA. Alternatively,trans-enoyl-coenzyme A reductase (Treponema denticola Ter) (E.C.1.3.1.86) generates butyryl-CoA from crotonyl-CoA and NADH. ButyrateCoA-transferase (R. eutropha Pct) (E.C. 2.8.3.1) generates butyrate andacetyl-CoA from butyryl-CoA and acetate. Aldehyde dehydrogenase (E. coliAdhE) (E.C. 1.2.1. {3,4}) generates butanal from butyrate and NADH.Alcohol dehydrogenase (E. coli adhE) (E.C. 1.1.1. {1,2}) generates1-butanol from butanal and NADH, NADPH. Production of 1-butanol isconferred by the engineered host cell by expression of the above enzymeactivities.

To create butanol-producing cells, host cells can be further engineeredto express acetyl-CoA acetyltransferase (atoB) from E. coli K12,β-hydroxybutyryl-CoA dehydrogenase from Butyrivibrio fibrisolvens,crotonase from Clostridium beijerinckii, butyryl CoA dehydrogenase fromClostridium beijerinckii, CoA-acylating aldehyde dehydrogenase (ALDH)from Cladosporium fulvum, and adhE encoding an aldehyde-alcoholdehydrogenase of Clostridium acetobutylicum (or homologs thereof).

Example 9 Engineered Organisms Producing Acrylate

Enoyl-CoA hydratase (E. coli paaF) (E.C. 4.2.1.17) converts3-hydroxypropionyl-CoA to acryloyl-CoA. Propionyl-CoA synthase (E.C.6.2.1.-, E.C. 4.2.1.- and E.C. 1.3.1.-) also converts3-hydroxypropionyl-CoA to acryloyl-CoA (AAL47820, SEQ ID NO:5). AcrylateCoA-transferase (R. eutropha pct) (E.C. 2.8.3.n) generatesacrylate+acetyl-CoA from acryloyl-CoA and acetate.

OTHER EMBODIMENTS

The examples have focused on Paracoccus. Nevertheless, the key conceptof using genetically engineering to confer production of carbon-basedproducts of interest to a methylotroph is extensible to othermethylotrophs such as other prokaryotes or eukaryotic single cellorganisms such as methylotrophic yeast. Alternatively, the energyconversion and/or carbon fixation pathways described in U.S. Pat. No.8,349,587 may be used to enhance or augment the methylotrophiccapability of an organism that is natively methylotrophic; U.S. Pat. No.8,349,587 is hereby incorporated by reference in its entirety.

Various aspects of the present invention may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing,” “involving,” andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

EQUIVALENTS

The present invention provides among other things novel methods andsystems for synthetic biology. While specific embodiments of the subjectinvention have been discussed, the above specification is illustrativeand not restrictive. Many variations of the invention will becomeapparent to those skilled in the art upon review of this specification.The full scope of the invention should be determined by reference to theclaims, along with their full scope of equivalents, and thespecification, along with such variations.

INCORPORATION BY REFERENCE

The Sequence Listing filed as an ASCII text file via EFS-Web (file name:“010401SequenceListing”; date of creation: Dec. 2, 2013; size: 25,461bytes) at the U.S. Patent and Trademark Office as the Receiving Officeis hereby incorporated by reference in its entirety.

All publications, patents and patent applications referenced in thisspecification are incorporated herein by reference in their entirety forall purposes to the same extent as if each individual publication,patent or patent application were specifically indicated to be soincorporated by reference.

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1. An engineered cell for producing a carbon-based product, comprisingan at least partially engineered carbon product biosynthetic pathwayintroduced into a methylotrophic organism, wherein said engineered cellis capable of converting a C1 compound into a carbon-based product ofinterest.
 2. The engineered cell of claim 1, wherein the methylotrophicorganism is capable of converting the C1 compound into a centralmetabolite.
 3. The engineered cell of claim 1 or 2, wherein the C1compound is soluble in water, such as formate, formic acid,formaldehyde, methanol, or any combination thereof.
 4. The engineeredcell of any one of claims 1-3, wherein the C1 compound is derived fromelectrolysis.
 5. The engineered cell of any one of claims 1-4, whereinsaid carbon-based product of interest is one or more of a sugar (forexample, glucose, fructose, sucrose, xylose, lactose, maltose, pentose,rhamnose, galactose or arabinose), sugar phosphate (for example,glucose-6-phosphate or fructose-6-phosphate), sugar alcohol (forexample, sorbitol), sugar derivative (for example, ascorbate), alcohol(for example, ethanol, propanol, isopropanol or butanol), fermentativeproduct (for example, ethanol, butanol, lactic acid, lactose oracetate), ethylene, propylene, 1-butene, 1,3-butadiene, acrylic acid,fatty acid (for example, O-cyclic fatty acid), fatty acid intermediateor derivative (for example, fatty acid alcohol, fatty acid ester,alkane, olegin or halogenated fatty acid), amino acid or intermediate(for example, lysine, glutamate, aspartate, shikimate, chorismate,phenylalanine, tyrosine, tryptophan), phenylpropanoid, isoprenoid (forexample, hemiterpene, monoterpene, sesquiterpene, triterpene,tetraterpene, polyterpene, isoprene, bisabolene, myrcene,amorpha-4,11-diene, farnesene, taxadiene, squalene, lanosterol,β-carotene, ζ-carotene, lycopene, phytoene, limonene, or polyisoprene),glycerol, 1,3-propanediol, 1,4-butanediol, 1,3-butadiene,polyhydroxyalkanoate, polyhydroxybutyrate, lysine, γ-valerolactone, andacrylate.
 6. The engineered cell of any one of claims 1-5, wherein whensaid carbon product biosynthetic pathway is for fatty acid biosynthesis,said carbon product biosynthetic pathway includes one or more of: fattyacid synthase, acetyl-CoA carboxylase, fatty-acyl-CoA reductase,aldehyde decarbonylase, lipase, thioesterase and acyl-CoA synthasepeptides; or when said carbon product biosynthetic pathway is forbranched chain fatty acid biosynthesis, said carbon product biosyntheticpathway includes one or more of: branched chain amino acidaminotransferase, branched chain α-ketoacid dehydrogenase, dihydrolipoyldehydrogenase, beta-ketoacyl-ACP synthase, crotonyl-CoA reductase,isobutyryl-CoA mutase, β-ketoacyl-ACP synthase I,trans-2,cis-3-decenoyl-ACP isomerase and trans-2-enoyl-ACP reductase II;or when said carbon product biosynthetic pathway is for fatty alcoholbiosynthesis, said carbon product biosynthetic pathway includes one ormore of: fatty alcohol forming acyl-CoA reductase, fatty alcohol formingacyl-CoA reductase, alcohol dehydrogenase and alcohol reductase; or whensaid carbon product biosynthetic pathway is for fatty esterbiosynthesis, said carbon product biosynthetic pathway includes one ormore of: alcohol O-acetyltransferase, wax synthase, fatty acid elongase,acyl-CoA reductase, acyltransferase, fatty acyl transferase,diacylglycerol acyltransferase, acyl-CoA was alcohol acyltransferase,bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase,and β-ketoacyl-ACP synthase I; or when said carbon product biosyntheticpathway is for alkane biosynthesis, said carbon product biosyntheticpathway includes one or more of: decarbonylase and terminal alcoholoxidoreductase; or when said carbon product biosynthetic pathway is forω-cyclic fatty acid biosynthesis, said carbon product biosyntheticpathway includes one or more of: 1-cyclohexenylcarbonyl CoA reductase,5-enopyruvylshikimate-3-phosphate synthase, acyl-CoA dehydrogenase,enoyl-(ACP) reductase, 2,4-dienoyl-CoA reductase, and acyl-CoAisomerase; or when said carbon product biosynthetic pathway is forhalogenated fatty acid biosynthesis, said carbon product biosyntheticpathway includes one or more of: fluorinase, nucleotide phosphorylase,fluorometabolite-specific aldolase, fluoroacetaldehyde dehydrogenase,and fluoroacetyl-CoA synthase; or when said carbon product biosyntheticpathway is the deoxylylulose 5-phosphate (DXP) isoprenoid pathway, saidcarbon product biosynthetic pathway includes one or more of:1-deoxy-D-xylulose-5-phosphate synthase, 1-deoxy-D-xylulose-5-phosphatereductoisomerase, 4-diphosphocytidyl-2C-methyl-D-erythritol synthase,4-diphosphocytidyl-2C-methyl-D-erythritol kinase, 2C-methyl-D-erythritol2,4-cyclodiphosphate synthase, (E)-4-hydroxy-3-methylbut-2-enyldiphosphate synthase, isopentyl/dimethylallyl diphosphate synthase and4-hydroxy-3-methylbut-2-enyl diphosphate reductase; or when said carbonproduct biosynthetic pathway is the mevalonate-dependent (MEV)isoprenoid pathway, said carbon product biosynthetic pathway includesone or more of: acetyl-CoA thiolase, HMG-CoA synthase, HMG-CoAreductase, mevalonate kinase, phosphomevalonate kinase, mevalonatepyrophosphate decarboxylase and isopentenyl pyrophosphate isomerase; orwhen said carbon product biosynthetic pathway is theglycerol/1,3-propanediol biosynthesis pathway, said carbon productbiosynthetic pathway includes one or more of: sn-glycerol-3-Pdehydrogenase, sn-glycerol-3-phosphatase, glycerol dehydratase and1,3-propanediol oxidoreductase; or when said carbon product biosyntheticpathway is the 1,4-butanediol/1,3-butadiene biosynthesis pathway, saidcarbon product biosynthetic pathway includes one or more of:succinyl-CoA dehydrogenase, 4-hydroxybutyrate dehydrogenase, aldehydedehydrogenase, 1,3-propanediol oxidoreductase and alcohol dehydratase;or when said carbon product biosynthetic pathway is thepolyhydroxybutyrate biosynthesis pathway, said carbon productbiosynthetic pathway includes one or more of: acetyl-CoA:acetyl-CoAC-acetyltransferase, (R)-3-hydroxyacyl-CoA:NADP⁺ oxidoreductase andpolyhydroxyalkanoate synthase; or when said carbon product biosyntheticpathway is the lysine biosynthesis pathway, said carbon productbiosynthetic pathway includes one or more of: aspartateaminotransferase, aspartate kinase, aspartate semialdehydedehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinatereductase, tetrahydrodipicolinate succinylase,N-succinyldiaminopimelate-aminotransferase, N-succinyl-L-diaminopimelatedesuccinylase, diaminopimelate epimerase, diaminopimelate decarboxylase,L,L-diaminopimelate aminotransferase, homocitrate synthase,homoaconitase, homoisocitrate dehydrogenase, 2-aminoadipatetransaminase, 2-aminoadipate reductase, aminoadipatesemialdehyde-glutamate reductase and lysine-2-oxoglutarate reductase; orwhen said carbon product biosynthetic pathway is the chorismatebiosynthesis pathway, said carbon product biosynthetic pathway includesone or more of: 2-dehydro-3-deoxyphosphoheptonate aldolase,3-dehydroquinate synthase, 3-dehydroquinate dehydratase, NADPH-dependentshikimate dehydrogenase, NAD(P)H-dependent shikimate dehydrogenase,shikimate kinase, 3-phosphoshikimate-1-carboxyvinyltransferase andchorismate synthase; or when said carbon product biosynthetic pathway isthe phenylalanine biosynthesis pathway, said carbon product biosyntheticpathway includes one or more of: chorismate mutase, prephenatedehydratase and phenylalanine transaminase; or when said carbon productbiosynthetic pathway is the tyrosine biosynthesis pathway, said carbonproduct biosynthetic pathway includes one or more of: chorismate mutase,prephenate dehydrogeanse and tyrosine aminotransferase; or when saidcarbon product biosynthetic pathway is the γ-valerolactone biosynthesispathway, said carbon product biosynthetic pathway includes one or moreof: propionyl-CoA synthase, beta-ketothiolase, acetoacetyl-CoAreductase, 3-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoAΔ-isomerase, 4-hydroxybutyryl-CoA transferase and 1,4-lactonase; or whensaid carbon product biosynthetic pathway is the butanol biosynthesispathway, said carbon product biosynthetic pathway includes one or moreof: beta-ketothiolase, acetoacetyl-CoA reductase, 3-hydroxybutyryl-CoAdehydrogenase, enoyl-CoA hydratase, butyryl-CoA dehydrogenase,trans-enoyl-coenzyme A reductase, butyrate CoA-transferase, aldehydedehydrogenase, alcohol dehydrogenase, acetyl-CoA acetyltransferase,β-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl CoAdehydrogenase, CoA-acylating aldehyde dehydrogenase and aldehyde-alcoholdehydrogenase; or when said carbon product biosynthetic pathway is theacrylate biosynthesis pathway, said carbon product biosynthetic pathwayincludes one or more of: enoyl-CoA hydratase, propionyl-CoA synthase andacrylate CoA-transferase.
 7. The engineered cell of claim 6, wherein the1-deoxy-D-xylulose-5-phosphate synthase is encoded by SEQ ID NO:1, or ahomolog thereof having at least 80% sequence identity; or theisopentenyl pyrophosphate isomerase is encoded by SEQ ID NO:2, or ahomolog thereof having at least 80% sequence identity.
 8. The engineeredcell of any one of claims 1-7, wherein when said carbon productbiosynthetic pathway is the isoprene biosynthesis pathway, said carbonproduct biosynthetic pathway includes isoprene synthase.
 9. Theengineered cell of claim 8, wherein the isoprene synthase is encoded bySEQ ID NO:3, or a homolog thereof having at least 80% sequence identity.10. The engineered cell of any one of claims 1-7, wherein when saidcarbon product biosynthetic pathway is the bisabolene biosynthesispathway, said carbon product biosynthetic pathway includesE-alpha-bisabolene synthase.
 11. The engineered cell of claim 10,wherein the E-alpha-bisabolene synthase is encoded by SEQ ID NO:4, or ahomolog thereof having at least 80% sequence identity.
 12. Theengineered cell of any one of claims 1-11, wherein the methylotrophicorganism is selected from the class Alphaproteobacterium.
 13. Theengineered cell of any one of claims 1-12, wherein the methylotrophicorganism is selected from the genus Paracoccus.
 14. The engineered cellof any one of claims 1-13, wherein the methylotrophic organism isParacoccus denitrificans, Paracoccus versutus or Paracoccuszeaxanthinifaciens.
 15. The engineered cell of any one of claims 1-14,further modified to have a less reduced growth rate on electrolyticallygenerated C1 compound relative to non-evolved methylotrophic organism,or a substantially similar or enhanced growth rate on electrolyticallygenerated C1 compound relative to non-electrolytically generated C1compound.
 16. The engineered cell of any one of claims 1-15, furtherevolved to have a less reduced growth rate on electrolytically generatedC1 compound relative to non-evolved methylotrophic organism, or asubstantially similar or enhanced growth rate on electrolyticallygenerated C1 compound relative to non-electrolytically generated C1compound.
 17. An evolved methylotrophic organism, having a less reducedgrowth rate on electrolytically generated C1 compound relative tonon-evolved methylotrophic organism, or having a substantially similaror enhanced growth rate on electrolytically generated C1 compoundrelative to non-electrolytically generated C1 compound.
 18. A method forselecting an evolved methylotrophic organism having improved growth on aC1 compound, comprising: incubating methylotrophic cells in a culturechamber with controlled temperature, cell concentration, and mediuminflow and outflow rates, wherein a medium inflow includes a C1compound; continuously monitoring a concentration of biomass in theculture chamber; and adjusting a flow rate of the C1 compound into theculture chamber so as to continually maintain an environment thatselects for an improved growth rate.
 19. The method of claim 18, furthercomprising adjusting the medium inflow to be more permissive of growthor more suppressive of growth, so as to provide an adaptive environmentto select for a fitness of the cells.
 20. The method of any one ofclaims 18-19, wherein the C1 compound is formate.
 21. The method of anyone of claims 18-20, wherein the C1 compound is electrolyticallygenerated.
 22. The method of any one of claims 18-21, wherein the C1compound is soluble in water.
 23. A method of introducing a conjugativeplasmid into methylotrophic host cells, comprising: incubating a mixtureof predetermined ratios of a donor culture and a recipient culture, attemperatures between 4° C. and 37° C. for between 1 and 48 hours,wherein the donor culture comprises a conjugal donor containing aconjugative plasmid having a first selectable trait, and the recipientculture comprises methylotrophic host cell having a second selectabletrait; and subjecting the incubated mixture to a dually selectivecondition where only plasmid-containing transconjugants that have boththe first selectable trait and the second selectable trait can grow,wherein the method does not include centrifugation or filtration of themixture or incubated mixture.
 24. The method of claim 23, wherein theconjugal donor is an E. coli strain such as E. coli S17-1, or an E. coliharboring plasmids such as pRK2013 or pRK2073, or any E. coli strainexpressing a tra operon capable of mobilizing plasmids containing anRP4-derived sequence.
 25. The method of claim 23 or 24, wherein theconjugal donor is in a different species or genus of the host cell. 26.The method of any one of claims 23-25, wherein the transconjugatedplasmid contains an RP4 or similar mob element.
 27. The method of anyone of claims 23-26, wherein the host cell is from the classAlphaproteobacterium.
 28. The method of any one of claims 23-27, whereinthe host cell is from the genus Paracoccus.
 29. The method of any one ofclaims 23-28, wherein the host cell is Paracoccus denitrificans,Paracoccus versutus or Paracoccus zeaxanthinifaciens.
 30. A compositionfor bacterial culture, formulated to provide formate as the sole sourceof C1 compound and to enhance growth of methylotrophic bacteria.
 31. Thecomposition of claim 30, comprising between 0 and 160 mM sodiumbicarbonate, between 0 and 16 mM sodium chloride, between 0 and 100 mMsodium nitrate, between 0 and 30 mM sodium thiosulfate, and initiallycontaining between 5 and 100 mM of a formate salt, such as sodiumformate and/or ammonium formate.
 32. The composition of any one ofclaims 30-31, comprising 100 mM sodium bicarbonate, 6 mM sodiumchloride, 6 mM sodium nitrate, 11 mM sodium thiosulfate, and 26 mMsodium formate or ammonium formate.
 33. The composition of any one ofclaims 30-33, further comprising a basal minimal medium.
 34. Thecomposition of claim 33, wherein the basal minimal medium is MOPSminimal medium, M9 minimal medium, R medium or M63 medium, or a mediumsubstantially similar thereto.
 35. A method for culturing methylotrophicbacteria, comprising incubating methylotrophic bacteria in thecomposition of any one of claims 30-34.
 36. A composition of bacterialculture, formulated to provide formate as the sole C1 compound and toenhance growth of methylotrophic bacteria in a fed-batch bioreactor. 37.The composition of claim 36, comprising a medium initially charged inthe fed-batch bioreactor which comprises R medium supplemented withbetween 1 and 100 micromolar sodium molybdate, between 10 and 1000nanomolar sodium selenite, between 0.01 to 1 mg/L of thiamine, andbetween 0.001 to 1 mg/L of cobalamin.
 38. The composition of claim 37,wherein the medium comprises between 5 and 20 micromolar sodiummolbydate, between 50 and 200 nanomolar sodium selenite, between 0.05 to2 mg/L of thiamine, between 0.01 and 0.2 mg/L cobalamin.
 39. Thecomposition of claim 37 or 38, further comprising a feed compositionsupplied to the fed-batch bioreactor comprising a formate salt atsupramolar concentration.
 40. The composition of claim 39, wherein theformate salt is ammonium formate and/or sodium formate.
 41. Thecomposition of claim 39 or 40, wherein feed composition furthercomprises a supramolar concentration of nitrate salt.
 42. Thecomposition of claim 41, wherein the nitrate salt is sodium nitrate. 43.The composition of any one of claims 39-42, wherein the nitrate salt andthe formate salt are provided in a molar ratio of 3.0:8 or lower.
 44. Amethod for culturing methylotrophic bacteria, comprising incubatingmethylotrophic bacteria in the composition of any one of claims 36-43 ina fed-batch bioreactor.
 45. The method of claim 44, wherein a volumetricrate of C1 feedstock consumption in the fed-batch reactor exceeds 1.5g*L⁻¹ hr⁻¹.
 46. The method of claim 45, wherein said incubating isconducted aerobically.
 47. The method of any one of claims 44-46,wherein said incubating is conducted in the presense of a nitrate saltas electron acceptor and formate salt as electron donor.
 48. The methodof claim 47, wherein a molar ratio of the nitrate salt to the formatesalt is kept below 3.2:8 in the fed-batch reactor.
 49. The method ofclaim 47 or 48, wherein the nitrate salt and the formate salt areprovided to the fed-batch bioreactor in a feed composition in supramolarconcentrations in a molar ratio of 3.0:8 or lower.
 50. The method of anyone of claims 47-49, wherein the formate salt is ammonium formate and/orsodium formate.
 51. The method of any one of claims 47-50, wherein thenitrate salt is sodium nitrate.